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Sample records for glycoprotein iib-iiia complex

  1. Contribution of inhibitory receptor glycoprotein iib / iiia in coronary angioplasty and acute coronary syndrome, about 152 patients

    International Nuclear Information System (INIS)

    Sellami, Walid

    2007-01-01

    The aim of our study was to evaluate the immediate results and long-term intake of anti-GP IIb / IIIa inhibitors for patients with acute coronary syndrome treated with coronary angioplasty. The use of anti-GP IIb / IIIa is a valid therapeutic option in patients with acute coronary syndrome with signs of severity and for patients undergoing complex angioplasty. Adverse effects of anti-GP IIb / IIIa can be seen to encourage vigilance and careful monitoring during the administration of these molecules and perfect knowledge of their pharmacological properties for appropriate use.

  2. Platelet glycoprotein IIb/IIIa polymorphism HPA-3 b/b is associated with increased risk of ischemic stroke in patients under 60 years of age.

    Science.gov (United States)

    Duan, Hao; Cai, Yan; Sun, Xiaojiang

    2012-01-01

    The role of genetic risk factors in ischemic stroke is unclear. Platelet glycoprotein IIb/IIIa (GpIIb-IIIa) has been implicated in the pathogenesis of ischemic stroke. We sought to evaluate the relationship between the GpIIb/IIIa complex gene polymorphism and ischemic stroke. We investigated the association of the GpIIb/IIIa complex gene polymorphism with stroke risk in 306 patients with acute ischemic stroke and 266 control subjects by determining the GpIIb and GpIIIa genotype from leukocyte DNA by polymerase chain reaction (PCR) followed by FokI and ScrFI digestion, respectively. Compared with controls, more patients presented with coronary heart disease, hypertension, smoking history, and diabetes. In addition, the patients had higher levels of cholesterol and glucose compared with the control subjects. All donors in the GpIIIa (n=572) group expressed the GpIIIa PlA1 (HPA-1 aa) phenotype. There were no significant differences between the HPA-3 genotype (GpIIb) patient distribution (aa=39.9%, ab=41.4%, bb=28.7%) and healthy control subjects (aa=36.1%, ab=35.0%, bb=28.9%) (P=0.580). Among study participants ischemic stroke >2-fold (P=0.008). The GpIIb Ile/Ser843 gene polymorphism is associated with ischemic stroke among young and middle-aged adults (ischemic stroke.

  3. An effect of glycoprotein IIb/IIIa inhibitors on the kinetics of red blood cells aggregation.

    Science.gov (United States)

    Sokolova, Irina A; Muravyov, Alexei V; Khokhlova, Maria D; Rikova, Sofya Yu; Lyubin, Evgeny V; Gafarova, Marina A; Skryabina, Maria N; Fedyanin, Angrey A; Kryukova, Darya V; Shahnazarov, Alexander A

    2014-01-01

    The reversible aggregation of red blood cells (RBCs) continues to be of the basic science and clinical interest. Recently it has been reported about a specific binding between fibrinogen and unknown erythrocyte glycoprotein receptors. The aim of this study was to investigate whether the red blood cell aggregation (RBCA) include the cell-cell interaction using the membrane receptors that bind such ligands as fibrinogen or fibronectin. To test this hypothesis the RBCs were incubated with monafram - the drug of the monoclonal antibodies against glycoprotein (GP) IIb/IIIa, with the GPIIb-IIIa receptor antagonist tirofiban, epifibatide and with the fibrinogen inhibiting peptide. It has been found that the RBC incubation with monafram resulted in a marked RBCA decrease mainly in persons with high level of aggregation. Another research session has shown that RBC incubation with fibronectin was accompanied by a significant RBCA rise. The monafram addition to red cell incubation medium resulted in a significant RBCA lowering. The cell incubation with tirofiban and epifibatide issued in RBCA decrease. The similar results were obtained when RBCs were incubated with the fibrinogen inhibiting peptide. Although monafram, tirofiban, eptifibatide and the fibrinogen inhibiting peptide were related to fibrinogen function they didn't inhibit RBCA completely. Therefore, under moderate and low red blood cell aggregation the cell binding is probably related to nonspecific mode. It seems evident that the specific and nonspecific modes of red blood cell aggregate formation could co-exist. Additional theoretical and experimental investigations in this area are needed.

  4. Safety and preliminary efficacy of one month glycoprotein IIb/IIIa inhibition with lefradafiban in patients with acute coronary syndromes without ST-elevation; a phase II study.

    OpenAIRE

    Akkerhuis, Martijn; Neuhaus, Karl; Wilcox, Robert; Vahanian, Alec; Boland, Jean; Hoffmann, J.; Baardman, Taco; Nehmiz, G.; Roth, U.; Klootwijk, Peter; Deckers, Jaap; Simoons, Maarten

    2000-01-01

    textabstractAIMS: Oral glycoprotein IIb/IIIa inhibitors might enhance the early benefit of an intravenous agent and prevent subsequent cardiac events in patients with acute coronary syndromes. We assessed the safety and preliminary efficacy of 1 month treatment with three dose levels of the oral GP IIb/IIIa blocker lefradafiban in patients with unstable angina or myocardial infarction without persistent ST elevation. METHODS: The Fibrinogen Receptor Occupancy STudy (FROST) was designed as a d...

  5. Bivalirudin versus heparin with or without glycoprotein IIb/IIIa inhibitors in patients with STEMI undergoing primary percutaneous coronary intervention

    DEFF Research Database (Denmark)

    Stone, Gregg W; Mehran, Roxana; Goldstein, Patrick

    2015-01-01

    bleeding and mortality rates, but higher acute stent thrombosis rates compared with heparin + a glycoprotein IIb/IIIa inhibitor (GPI). Subsequent changes in primary PCI, including the use of potent P2Y12 inhibitors, frequent radial intervention, and pre-hospital medication administration, were incorporated...... approach was used in 21.3% of patients, prasugrel/ticagrelor was used in 18.1% of patients, and GPI was used in 84.8% of the control group. Bivalirudin compared with heparin ± GPI resulted in reduced 30-day rates of major bleeding (4.2% vs. 7.8%; relative risk [RR]: 0.53; 95% confidence interval [CI]: 0...

  6. Genomic clone encoding the α chain of the OKM1, LFA-1, and platelet glycoprotein IIb-IIIa molecules

    International Nuclear Information System (INIS)

    Cosgrove, L.J.; Sandrin, M.S.; Rajasekariah, P.; McKenzie, I.F.C.

    1986-01-01

    LFA-1, an antigen involved in cytolytic T lymphocyte-mediated killing, and Mac-1, the receptor for complement component C3bi, constitute a family of structurally and functionally related cell surface glycoproteins involved in cellular interactions. In both mouse and man, Mac-1 (OKM1) and LFA-1 share a common 95-kDa β subunit but are distinguished by their α chains, which have different cellular distributions, apparent molecular masses (165 and 177 kDa, respectively), and peptide maps. The authors report the isolation of a genomic clone from a human genomic library that on transfection into mouse fibroblasts produced a molecule(s) reactive with monoclonal antibodies to OKM1, to LFA-1, and to platelet glycoprotein IIb-IIIa. This gene was cloned by several cycles of transfection of L cells with a human genomic library cloned in λ phase Charon 4A and subsequent rescue of the λ phage. Transfection with the purified recombinant λ DNA yielded a transfectant that expressed the three human α chains of OKM1, LFA-1, and glycoprotein IIb-IIIa, presumably in association with the murine β chain

  7. Antiplatelet activity of L-sulforaphane by regulation of platelet activation factors, glycoprotein IIb/IIIa and thromboxane A2.

    Science.gov (United States)

    Oh, Chung-Hun; Shin, Jang-In; Mo, Sang Joon; Yun, Sung-Jo; Kim, Sung-Hoon; Rhee, Yun-Hee

    2013-07-01

    L-sulforaphane was identified as an anticarcinogen that could produce quinine reductase and a phase II detoxification enzyme. In recent decades, multi-effects of L-sulforaphane may have been investigated, but, to the authors' knowledge, the antiplatelet activation of L-sulforaphane has not been studied yet.In this study, 2 μg/ml of collagen, 50 μg/ml of ADP and 5 μg/ml of thrombin were used for platelet aggregations with or without L-sulforaphane. L-sulforaphane inhibited the platelet aggregation dose-dependently. Among these platelet activators, collagen was most inhibited by L-sulforaphane, which markedly decreased collagen-induced glycoprotein IIb/IIIa activation and thromboxane A2 (TxA2) formation in vitro. L-sulforaphane also reduced the collagen and epinephrine-induced pulmonary embolism, but did not affect prothrombin time (PT) in vivo. This finding demonstrated that L-sulforaphane inhibited the platelet activation through an intrinsic pathway.L-sulforaphane had a beneficial effect on various pathophysiological pathways of the collagen-induced platelet aggregation and thrombus formation as a selective inhibition of cyclooxygenase and glycoprotein IIb/IIIa antagonist. Thus, we recommend L-sulforaphane as a potential antithrombotic drug.

  8. Safety and preliminary efficacy of one month glycoprotein IIb/IIIa inhibition with lefradafiban in patients with acute coronary syndromes without ST-elevation; a phase II study.

    NARCIS (Netherlands)

    K.M. Akkerhuis (Martijn); K.L. Neuhaus (Karl); R.G. Wilcox (Robert); A. Vahanian (Alec); J-L. Boland (Jean); J. Hoffmann; T. Baardman (Taco); G. Nehmiz; U. Roth; A.P.J. Klootwijk (Peter); J.W. Deckers (Jaap); M.L. Simoons (Maarten)

    2000-01-01

    textabstractAIMS: Oral glycoprotein IIb/IIIa inhibitors might enhance the early benefit of an intravenous agent and prevent subsequent cardiac events in patients with acute coronary syndromes. We assessed the safety and preliminary efficacy of 1 month treatment with three dose levels of the oral GP

  9. Initial experience with a new femoral artery closure device following percutaneous coronary intervention with glycoprotein IIb/IIIa inhibition.

    Science.gov (United States)

    Ruygrok, Peter N; Ormiston, John A; Stewart, James T; Webster, Mark W I; El Jack, Seif; Simpson-Plaumann, Stephanie; Kay, I Patrick; Chou, Tony M

    2005-10-01

    The aim of the study was to determine the safety and efficacy of a novel femoral artery closure device (StarClose, Abbott Vascular Devices, Redwood City, CA) following percutaneous coronary intervention employing aspirin, heparin, and glycoprotein (GP) IIb/IIIa inhibition. A prospective nonrandomized single-center pilot study of the StarClose device included a subset of patients undergoing percutaneous coronary intervention utilizing GP IIb/IIIa inhibitors. Those that fulfilled the inclusion criteria (age < 80, no periprocedural haematoma, puncture above the superficial femoral and profunda femoralis artery bifurcation, no significant femoral artery disease) underwent closure of the femoral artery puncture site with a StarClose device immediately on completion of the procedure. Time to hemostasis (TTH), bleeding, mobilization, and short-term clinical follow-up data were collected, and an ultrasound scan of the femoral artery was performed 2 weeks later. Twenty-five patients were recruited, of whom 23 underwent percutaneous coronary intervention (PCI). Their mean age was 58 +/- 12 years, 84% were male, and 63% had unstable angina. All were on aspirin 100-150 mg daily and all PCI patients received i.v. heparin 4-10,000 units at commencement of the procedure and clopidogrel 600 mg on completion. Two patients were on a tirofiban infusion and 23 received a double bolus of eptifibatide, each 0.18 mg/kg, separated by 10 min. The procedural success was 100% and device success 23/25 (92%), with 1 failure due to technical error. The median device delivery time was 36 sec (range, 11-178) and median TTH 37 sec (range, 10-509 sec). There were no major adverse events. In 10 patients, a moderate amount of tract ooze required a short period of adjunctive manual compression. Follow-up ultrasound femoral artery scans revealed no compromise of the vessel lumen. Femoral artery closure with the device following coronary angiography and intervention using glycoprotein IIb/IIIa receptor

  10. Combining low-molecular-weight heparin and glycoprotein IIb/IIIa antagonists for the treatment of acute coronary syndromes: the NICE 3 story. National Investigators Collaborating on Enoxaparin.

    Science.gov (United States)

    Ferguson, J J

    2000-12-01

    In recent years, a number of new therapies have become available that have significantly improved the care of patients with acute coronary syndromes (ACS). A number of studies have documented the significant superiority of the low-molecular-weight heparin (LMWH), enoxaparin, over unfractionated heparin (UFH) in the treatment of ACS. However, there are insufficient data regarding the safety of using LMWH in combination with glycoprotein (GP) IIb/IIIa antagonists. The NICE 3 study is an open-labeled, non-randomized, observational study examining the feasibility, safety, and preliminary efficacy of the LMWH, enoxaparin, in combination with each of the three commercially available GP IIb/IIIa antagonists in ACS patients. Approximately 600 patients at 45 clinical centers in North America will be divided between the three arms of the trial, representing each of the three GP IIb/IIIa antagonists. Once therapy is initiated, patients will receive no UFH (unless they require a coronary artery bypass graft) and will continue treatment even if invasive cath lab procedures are necessary. NICE 3 builds on the previous experiences of NICE 1 and NICE 4 with LMWH in the cath lab (with or without GP IIb/IIIa antagonists) to more aggressively extend the use of LMWHs into the medical management of ACS patients.

  11. Procedural and clinical outcomes after use of the glycoprotein IIb/IIIa inhibitor abciximab for saphenous vein graft interventions

    International Nuclear Information System (INIS)

    Harskamp, Ralf E.; Hoedemaker, Niels; Newby, L. Kristin; Woudstra, Pier; Grundeken, Maik J.; Beijk, Marcel A.; Piek, Jan J.; Tijssen, Jan G.; Mehta, Rajendra H.; Winter, Robbert J. de

    2016-01-01

    Background: Percutaneous coronary intervention (PCI) of saphenous vein grafts (SVG) poses a high-risk for distal coronary thromboembolic events. Glycoprotein IIb/IIIa inhibitors are frequently used in hope of reducing the impact of this, although the safety and efficacy of these drugs to improve outcomes in this setting are understudied. Methods: Patients were included if they had prior coronary artery bypass surgery and subsequently underwent PCI of ≥ 1 SVG graft at a Dutch academic center between 1997 and 2008. These patients were matched 1:1 based on peri-procedural use of abciximab using a propensity-score matching algorithm based on 17 variables. Conditional logistic regression and Cox regression stratified on matched pairs were performed to evaluate the association between abciximab use and MACCE (the composite measure of mortality, myocardial infarction, stroke and repeat revascularization) at 30 days and up to 1 year. Results: The composite of 30-day MACCE occurred in 18 patients (15.3%) in the abciximab group and 16 patients (13.6%) in the propensity matched control group (OR: 1.13, 95% CI: 0.57–2.21, p = 0.73). At 1-year follow-up, MACCE rates were also similar (32.5% vs. 33.9%, HR: 0.97, 95% CI: 0.59–1.59). Major bleeding (BARC types 3a–c) was higher in the abciximab group (11.9% vs. 4.2%, OR: 2.80, 95% CI: 1.01–7.77). Ischemic outcomes did not differ among patients with acute coronary syndromes. Conclusion: The use of intravenous abciximab was not associated with improved clinical outcomes up to 1-year among patients undergoing SVG PCI, but was related to more bleeding. - Highlights: • PCI of SVG poses a high-risk for distal coronary thromboembolic events. • Glycoprotein IIb/IIIa inhibitors are frequently used in an attempt to reduce this risk. • We evaluated the safety and efficacy of abciximab (a glycoprotein IIb/IIIa inhibitor) using a propensity-score matched analysis of 236 patients at a large academic medical center. • Thirty

  12. Procedural and clinical outcomes after use of the glycoprotein IIb/IIIa inhibitor abciximab for saphenous vein graft interventions

    Energy Technology Data Exchange (ETDEWEB)

    Harskamp, Ralf E., E-mail: r.e.harskamp@gmail.com [Academic Medical Center–University of Amsterdam, Amsterdam (Netherlands); VU University Medical Center, Amsterdam (Netherlands); Duke Clinical Research Institute, Durham, NC (United States); Hoedemaker, Niels [Academic Medical Center–University of Amsterdam, Amsterdam (Netherlands); Newby, L. Kristin [Duke Clinical Research Institute, Durham, NC (United States); Woudstra, Pier; Grundeken, Maik J.; Beijk, Marcel A.; Piek, Jan J.; Tijssen, Jan G. [Academic Medical Center–University of Amsterdam, Amsterdam (Netherlands); Mehta, Rajendra H. [Duke Clinical Research Institute, Durham, NC (United States); Winter, Robbert J. de [Academic Medical Center–University of Amsterdam, Amsterdam (Netherlands)

    2016-01-15

    Background: Percutaneous coronary intervention (PCI) of saphenous vein grafts (SVG) poses a high-risk for distal coronary thromboembolic events. Glycoprotein IIb/IIIa inhibitors are frequently used in hope of reducing the impact of this, although the safety and efficacy of these drugs to improve outcomes in this setting are understudied. Methods: Patients were included if they had prior coronary artery bypass surgery and subsequently underwent PCI of ≥ 1 SVG graft at a Dutch academic center between 1997 and 2008. These patients were matched 1:1 based on peri-procedural use of abciximab using a propensity-score matching algorithm based on 17 variables. Conditional logistic regression and Cox regression stratified on matched pairs were performed to evaluate the association between abciximab use and MACCE (the composite measure of mortality, myocardial infarction, stroke and repeat revascularization) at 30 days and up to 1 year. Results: The composite of 30-day MACCE occurred in 18 patients (15.3%) in the abciximab group and 16 patients (13.6%) in the propensity matched control group (OR: 1.13, 95% CI: 0.57–2.21, p = 0.73). At 1-year follow-up, MACCE rates were also similar (32.5% vs. 33.9%, HR: 0.97, 95% CI: 0.59–1.59). Major bleeding (BARC types 3a–c) was higher in the abciximab group (11.9% vs. 4.2%, OR: 2.80, 95% CI: 1.01–7.77). Ischemic outcomes did not differ among patients with acute coronary syndromes. Conclusion: The use of intravenous abciximab was not associated with improved clinical outcomes up to 1-year among patients undergoing SVG PCI, but was related to more bleeding. - Highlights: • PCI of SVG poses a high-risk for distal coronary thromboembolic events. • Glycoprotein IIb/IIIa inhibitors are frequently used in an attempt to reduce this risk. • We evaluated the safety and efficacy of abciximab (a glycoprotein IIb/IIIa inhibitor) using a propensity-score matched analysis of 236 patients at a large academic medical center. • Thirty

  13. Design of potent and specific integrin antagonists. Peptide antagonists with high specificity for glycoprotein IIb-IIIa.

    Science.gov (United States)

    Scarborough, R M; Naughton, M A; Teng, W; Rose, J W; Phillips, D R; Nannizzi, L; Arfsten, A; Campbell, A M; Charo, I F

    1993-01-15

    Members of the snake venon-derived, "disintegrin" peptide family containing the Arg-Gly-Asp (RGD) amino acid sequence are among the most potent inhibitors of the binding of adhesive proteins to platelet glycoprotein (GP) IIb-IIIa. However, GPIIb-IIIa antagonists containing the RGD sequence are not integrin specific and inhibit the adhesive functions of many other RGD-dependent integrins. The single disintegrin peptide, barbourin, containing a conservative amino acid substitution of Lys (K) for Arg (R) in the RGD sequence, is however, highly specific for GPIIb-IIIa. Using this information we have tested the hypothesis that both structural and conformational elements of barbourin are important for its high affinity and selectivity for platelet GPIIb-IIIa by synthesizing a series of conformationally constrained, disulfide-bridged peptides containing the KGD amino acid sequence. Incorporation of the KGD sequence into a cyclic peptide template, followed by systematic optimization of the cyclic ring size, optimization of secondary hydrophobic binding site interactions, and the derivatization of the lysyl side chain functionality of the KGD sequence has resulted in peptide analogs which display inhibitory potency and GPIIb-IIIa selectivity comparable to that of barbourin. This study demonstrates that the specificity and potency of the disintegrin family of antagonists, in particular barbourin, can be mimicked by small, conformationally restrained peptides.

  14. Safety and preliminary efficacy of one month glycoprotein IIb/IIIa inhibition with lefradafiban in patients with acute coronary syndromes without ST-elevation; a phase II study.

    Science.gov (United States)

    Akkerhuis, K M; Neuhaus, K L; Wilcox, R G; Vahanian, A; Boland, J L; Hoffmann, J; Baardman, T; Nehmiz, G; Roth, U; Klootwijk, A P; Deckers, J W; Simoons, M L

    2000-12-01

    Oral glycoprotein IIb/IIIa inhibitors might enhance the early benefit of an intravenous agent and prevent subsequent cardiac events in patients with acute coronary syndromes. We assessed the safety and preliminary efficacy of 1 month treatment with three dose levels of the oral GP IIb/IIIa blocker lefradafiban in patients with unstable angina or myocardial infarction without persistent ST elevation. The Fibrinogen Receptor Occupancy STudy (FROST) was designed as a dose-escalation trial with 20, 30 and 45 mg lefradafiban t.i.d. or placebo. Five hundred and thirty-one patients were randomized in a 3:1 ratio to lefradafiban or placebo in a double-blind manner. Efficacy was assessed by the incidence of death, myocardial infarction, coronary revascularization and recurrent angina. Safety was evaluated by the occurrence of bleeding classified according to the TIMI criteria and by measuring clinical laboratory parameters. There was a trend towards a reduction in cardiac events with lefradafiban 30 mg when compared with placebo and lefradafiban 20 mg. The benefit was particularly apparent in patients with a positive (> or = O.1 ng. ml(-1)) troponin I test at baseline and less so in those with a negative test result. In patients receiving lefradafiban, the cardiac event rate decreased with increasing minimal levels of fibrinogen receptor occupancy. There was a dose-dependent increase in the incidence of bleeding: the composite of major or minor bleeding occurred in 1% of placebo patients, 5% of patients receiving lefradafiban 20 mg and in 7% of patients receiving 30 mg, with an excessive risk (15%) in the 45 mg group which resulted in early discontinuation of this dose level. Gingival and arterial or venous puncture site bleedings were most common and accounted for more than 60% of all haemorrhagic events. There was an increased incidence of neutropenia (neutrophils <1. 5 x 10(9)/l) in the lefradafiban groups (5.2% vs 1.5% in the placebo group), which did not result from

  15. Scintigraphic detection of acute experimental endocarditis with the technetium-99m labelled glycoprotein IIb/IIIa receptor antagonist DMP444

    Energy Technology Data Exchange (ETDEWEB)

    Oyen, W.J.G.; Boerman, O.C.; Corstens, F.H.M. [Department of Nuclear Medicine, University Hospital Nijmegen, Nijmegen (Netherlands); Brouwers, F.M. [Department of Nuclear Medicine, University Hospital Nijmegen, Nijmegen (Netherlands); Department of Internal Medicine, University Hospital Nijmegen, Nijmegen (Netherlands); Barrett, J.A. [DuPont Pharmaceutical Company, Radiopharmaceutical Division, North Billerica, MA (United States); Verheugt, F.W.A. [Department of Cardiology, University Hospital Nijmegen, Nijmegen (Netherlands); Ruiter, D.J. [Department of Pathology, University Hospital Nijmegen, Nijmegen (Netherlands); Meer, J.W.M. van der [Department of Internal Medicine, University Hospital Nijmegen, Nijmegen (Netherlands)

    2000-04-01

    Bacterial endocarditis is an important clinical problem that may result in persistent bacteraemia and irreversible cardiac damage. Since endocarditis is characterized by aggregation of activated platelets, fibrin and bacteria, we studied DMP444, a technetium-99m labelled high-affinity antagonist of the GP IIb/IIIa receptor that is expressed on activated platelets. In seven Beagle dogs (11-15 kg), the left ventricle was catheterized via the right carotid artery. One hour later, 5 x 10{sup 7} colony forming units of Staphylococcus aureus were injected intracardially. Half an hour later, the catheter was removed. Two extra dogs underwent a complete sham procedure. One day after the intervention, five infected and the two non-infected dogs were injected with 37 MBq/kg {sup 99m}Tc-DMP444 and two infected dogs with 37 MBq/kg {sup 99m}Tc-IgG (used as a non-specific control agent) and imaged up to 4 h after injection. Samples were obtained for tissue counting, microbiology and histology. From 1 to 2 h post injection onward, there was clear focal accumulation of DMP444 in the aortic valve region when endocarditis was present, and this accumulation increased with time. The non-infected and the {sup 99m}Tc-IgG injected dogs showed only persisting blood pool activity without any focal abnormality. At 4 h post injection, the in vivo valve-to-blood pool ratios were 1.87{+-}0.18 in endocarditis, 1.01{+-}0.05 in non-infected controls and 1.09{+-}0.02 in {sup 99m}Tc-IgG injected dogs (P<0.05). It is concluded that targeting activated platelets with the {sup 99m}Tc-labelled GP IIb/IIIa antagonist DMP444 allows a final diagnosis of experimental bacterial endocarditis within 4 h owing to high, specific and fast in vivo uptake. (orig.)

  16. Scintigraphic detection of acute experimental endocarditis with the technetium-99m labelled glycoprotein IIb/IIIa receptor antagonist DMP444

    International Nuclear Information System (INIS)

    Oyen, W.J.G.; Boerman, O.C.; Corstens, F.H.M.; Brouwers, F.M.; Barrett, J.A.; Verheugt, F.W.A.; Ruiter, D.J.; Meer, J.W.M. van der

    2000-01-01

    Bacterial endocarditis is an important clinical problem that may result in persistent bacteraemia and irreversible cardiac damage. Since endocarditis is characterized by aggregation of activated platelets, fibrin and bacteria, we studied DMP444, a technetium-99m labelled high-affinity antagonist of the GP IIb/IIIa receptor that is expressed on activated platelets. In seven Beagle dogs (11-15 kg), the left ventricle was catheterized via the right carotid artery. One hour later, 5 x 10 7 colony forming units of Staphylococcus aureus were injected intracardially. Half an hour later, the catheter was removed. Two extra dogs underwent a complete sham procedure. One day after the intervention, five infected and the two non-infected dogs were injected with 37 MBq/kg 99m Tc-DMP444 and two infected dogs with 37 MBq/kg 99m Tc-IgG (used as a non-specific control agent) and imaged up to 4 h after injection. Samples were obtained for tissue counting, microbiology and histology. From 1 to 2 h post injection onward, there was clear focal accumulation of DMP444 in the aortic valve region when endocarditis was present, and this accumulation increased with time. The non-infected and the 99m Tc-IgG injected dogs showed only persisting blood pool activity without any focal abnormality. At 4 h post injection, the in vivo valve-to-blood pool ratios were 1.87±0.18 in endocarditis, 1.01±0.05 in non-infected controls and 1.09±0.02 in 99m Tc-IgG injected dogs (P 99m Tc-labelled GP IIb/IIIa antagonist DMP444 allows a final diagnosis of experimental bacterial endocarditis within 4 h owing to high, specific and fast in vivo uptake. (orig.)

  17. Local Intracoronary Infusion of Glycoprotein IIb/IIIa Inhibitors via a Perfusion Catheter versus Intracoronary Guiding Catheter Injection during Primary Percutaneous Coronary Intervention: A Pilot Observational Study.

    Science.gov (United States)

    Zaki, Tarek; Labib, Salwa; El-Abbady, Maged; El-Kilany, Wael; Mortada, Ayman; Rashid, Tarek; Ragy, Hany; El-Itreby, Adel; Nammas, Wail

    2017-05-01

    Glycoprotein IIb IIIa inhibitors improved short- and long-term outcome when added to primary percutaneous coronary intervention (PPCI) in patients with ST-segment-elevation myocardial infarction (STEMI). We hypothesized that intracoronary eptifibatide infusion via a perfusion catheter improves angiographic and clinical outcome of patients with STEMI undergoing PPCI, versus conventional intracoronary bolus injection. Prospectively, we enrolled 80 patients with acute STEMI and thrombolysis in myocardial infarction (TIMI) thrombus grade ≥ 2. Patients were assigned to receive eptifibatide (180 μg) either via a dedicated coronary perfusion catheter (ClearWay TM ) during PPCI (group I), or guiding catheter (group II). Assessment of TIMI thrombus grade, TIMI flow grade, and TIMI myocardial perfusion (TMP) grade was performed both at baseline and post- procedurally. The primary 'angiographic' endpoint was final TMP grade 0/1. The primary 'clinical' endpoint was a composite of cardiac death, non-fatal re-infarction, target vessel revascularization, and recurrent ischemia at 30-day follow-up. Mean age was 52.3 ± 8.9 years (17.5% females). Clearance of visible thrombus (TIMI thrombus grade 0) at final angiogram was more frequent in group I. Additionally, both final TIMI flow grade 3 and final TMP grade 3 occurred more frequently in group I. The primary angiographic endpoint was more frequent in group II versus group I (17.5% versus 0%, respectively, p = 0.001). The primary clinical endpoint was more frequent in group II (20% versus 0%, respectively, p = 0.003). In patients with STEMI, intracoronary eptifibatide infusion via a perfusion catheter during PPCI improved immediate angiographic outcome, and reduced clinical events at 30-day follow-up, versus bolus injection via the guiding catheter.

  18. Frequency of anti-glycoprotein Ia/IIa (anti-HPA-5b,-5a and anti-glycoprotein IIb/IIIa (anti-HPA-1a,-3a,-4a alloantibodies in multiparous women of African descent

    Directory of Open Access Journals (Sweden)

    Zaccheaus A Jeremiah

    2010-05-01

    Full Text Available Zaccheaus A Jeremiah1, Justina E Oburu2, Osaro Erhabor1, Fiekumo I Buseri1, Teddy C Adias31Haematology and Blood Transfusion Science Unit, Department of Medical Laboratory Sciences, College of Health Sciences, Niger Delta University, Wilberforce Island, Nigeria; 2Department of Haematology and Blood Transfusion, University of Port Harcourt Teaching Hospital, Port Harcourt, Nigeria; 3Rivers State University of Science and Technology, Port Harcourt, NigeriaBackground: Human platelet antibodies are often implicated in some disease conditions, such as neonatal alloimmune thrombocytopenia (NAIT, idiopathic thrombocytopenic purpura (ITP and platelet refractoriness. The frequencies of these alloantibodies have not been reported in Nigeria and West Africa.Methods: Screening for allontibodies to human platelet antigens (HPA was undertaken using the GTI PakPlus® qualitative solid phase ELISA reagent. Platelet count was done using the ICSH approved procedure using 1% ammonium oxalate reagent.Study design: A cross-section of apparently healthy adult Nigerian multiparous non-pregnant women, who were staff of a tertiary health facility in the Niger Delta, Nigeria, were screened for alloantibodies to human platelet antigens.Results: Of the one hundred (100 women screened, the prevalence of anti-glycoprotein IIb/IIIa (anti-HPA-Ia,-3a,-4a was zero percent (0%, anti-glycoprotein Ia/IIa (anti-HPA-5b accounted for 30% of results, while anti-glycoprotein Ia/IIa (anti-HPA-5a was 18%. Parity was found to exert significant influence on the development to HPA antibodies (Fisher’s Exact Test = 11.683, P < 0.05; 13.577, P < 0.01. The platelet count of the women did not appear to exert any influence on the development of the antibodies (P > 0.05.Conclusion: This study has observed a high prevalence of anti-HPA-5b in our sample population. The prevalence of alloantibodies to HPA antigens was found to associate strongly with parity. These results indicate that there is a

  19. PLA2 polymorphism of platelet glycoprotein IIb/IIIa but not Factor V Leiden and prothrombin G20210A polymorphisms is associated with venous thromboembolism and more recurrent events in central Iran.

    Science.gov (United States)

    Pourgheysari, Batoul; Boroujeni, Hamid Rouhi; Hasheminia, Ali Mohammad; Drees, Fatima

    2013-07-01

    Inherited thrombophilic gene polymorphisms have been linked to the pathogenesis of venous thromboembolism (VTE). As there are very limited data of these polymorphisms in the Iranian population, we aimed to investigate the correlation between them and VTE in central Iran. Seventy-two unrelated VTE patients and 306 healthy control individuals were recruited for the study. Genotyping from venous blood with EDTA for the factor V Leiden (FVL), prothrombin (FII) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T and PLA2 polymorphism of platelet glycoprotein IIb/IIIa were undertaken by PCR-restriction fragment length polymorphism (PCR-RFLP). A total of 57 investigated polymorphisms with a mean of 0.79 per individual and 151 with a mean of 0.49 were found in patients and controls, respectively (Pgenetic risk factor (P=0.007) and more recurrent events occurred in such patients. Patients with PLA2 polymorphism had more recurrent events than the other patients (P=0.02). Patients with more than one genetic risk factor and recurrent events were younger. The prevalence of these polymorphisms is different from some previously published data in other populations, but is consistent with some others. Higher prevalence of PLA2 polymorphism of GPIIa/IIIb in VTE patients is indicative of the impact of this polymorphism in the pathogenesis of VTE in this population. Because of the impact of coinheritance on the recurrence and the age of occurrence, such patients may need to be managed differently.

  20. Efeito protetor de antagonista das gliproteínas IIb/IIa nas alterações hepáticas e pulmonares secundárias à isquemia e reperfusão do fígado em ratos Protective effects of an inhibitor of glycoprotein IIb/IIIa in the hepatic and pulmonary disturbances secondary to ischemia and reperfusion injury of rat’s liver

    Directory of Open Access Journals (Sweden)

    Leonardo F. Canedo

    2007-09-01

    Full Text Available RACIONAL: A lesão de isquemia e reperfusão hepática é um evento comum e responsável por considerável morbidade e mortalidade. OBJETIVO: Avaliar efeitos de inibidor da glicoproteína IIb/IIIa, cloridrato de tirofiban, nas alterações hepáticas e pulmonares da lesão de isquemia e reperfusão de fígado de ratos. MÉTODO: Vinte e três ratos Wistar divididos em três grupos: laparotomia (n = 6, isquemia e reperfusão que receberam solução fisiológica (n = 8, e submetidos a isquemia e reperfusão e tratados com o cloridrato de tirofiban (n = 9. Foram realizadas dosagens das aminotransferases e análise histológica hepática. Avaliação pulmonar foi realizada pelo teste do azul de Evans e pela dosagem tecidual da mieloperoxidase no parênquima pulmonar. A oxidação e fosforilação mitocondrial das células hepáticas também foram avaliadas. RESULTADOS: O grupo tratado com cloridrato de tirofiban apresentou menores níveis de aminotransferases, assim como alterações histológicas menos intensas. Avaliação pulmonar demonstrou diminuição no teste de azul de Evans no grupo tratado com cloridrato de tirofiban. Grupo tratado com cloridrato de tirofiban apresentou aumento significativo do estado 3 da respiração mitocondrial e das relações adenosina difosfato utilizado para fosforilação sobre o oxigênio consumido na reação e de coeficiente respiratório. CONCLUSÕES: O uso do cloridrato de tirofiban exerceu papel protetor da lesão hepática de isquemia e reperfusão e impediu o aumento da permeabilidade vascular secundária à lesão de reperfusão hepática.BACKGROUND Hepatic ischemia-reperfusion injury is responsible for a considerable morbidity and mortality. Aim - To evaluate the effect of a platelet glycoprotein IIb/IIIa receptor inhibitor (tirofiban on hepatic and pulmonary disturbances associated with hepatic ischemia-reperfusion injury. METHODS: Twenty-three Wistar rats divided in three groups: rats sham-operated (n

  1. Increased risk for vascular complications due to GP IIb/IIIa-antagonists in patients with cardiogenic shock supported by intraaortic balloon pump (IABP

    Directory of Open Access Journals (Sweden)

    Jens Röther

    2015-08-01

    Conclusion: Vascular events with the use of IABP are common but in our study, not significantly associated with a higher mortality. Treatment with GP IIb/IIIa-antagonists is associated with a higher risk of vascular events.

  2. Long-term treatment with a platelet glycoprotein-receptor antagonist after percutaneous coronary revascularization. EXCITE Trial Investigators. Evaluation of Oral Xemilofiban in Controlling Thrombotic Events.

    NARCIS (Netherlands)

    P.W.J.C. Serruys (Patrick); M. Knudtson; G.A. van Es (Gerrit Anne); G.C. Timmis; C. van der Zwaan (Coen); J. Kleiman; J. Gong (Jianjian); E.B. Roecker; R. Dreiling; J.C. Alexander (John); R.J. Anders (Robert); W.W. O'Neill (William)

    2000-01-01

    textabstractBACKGROUND: When administered intravenously at the time of percutaneous coronary revascularization, glycoprotein IIb/IIIa receptor antagonists decrease the incidence of death and nonfatal myocardial infarction and the need for urgent revascularization. We hypothesized that long-term

  3. [Immunization against a new, infrequent alloantigen (Iy) on the platelet glycoprotein Ib/IX as a cause of a serious case of neonatal alloimmune thrombocytopenia].

    Science.gov (United States)

    Kiefel, V; Vicariot, M; Breitfeld, C; Giptner, A; Schlüter, C; Böhringer, M; Santoso, S; Kroll, H; Mueller-Eckhardt, C

    1994-01-01

    Neonatal alloimmune thrombocytopenia is the consequence of maternal alloimmunization against platelet-specific alloantigens, usually PIA1 or Br(a). The clinical picture is characterized by signs of haemorrhagic diathesis as a result of marked thrombocytopenia. In the last years, rare cases of immunization against 'low-frequency' or 'private' platelet alloantigens on the platelet glycoprotein (GP) complex IIb/IIIa have been found. This is the first report on NAIT due to maternal immunization against a low-frequency platelet alloantigen ('Iy') localized on platelet GP Ib/IX.

  4. Inhibition of tumor cell-induced platelet aggregation and lung metastasis by the oral GpIIb/IIIa antagonist XV454.

    Science.gov (United States)

    Amirkhosravi, Ali; Mousa, Shaker A; Amaya, Mildred; Blaydes, Susan; Desai, Hina; Meyer, Todd; Francis, John L

    2003-09-01

    Platelets are known to play a role in blood borne metastasis. Previous experimental studies have suggested that platelet GpIIb/IIIa may be a therapeutic target. However, the need for intravenous administration limits the potential application of current GpIIb/IIIa inhibitors to cancer therapy. The aim of the present study was to assess the efficacy of a novel, non-peptide oral GpIIb/IIIa antagonist (XV454) on tumor cell-induced platelet aggregation in vivo and on experimental metastasis. A Lewis lung carcinoma (LL2) mouse model of experimental metastasis was used in this study. XV454 (100 micro g) was administered intravenously (via tail vein) or orally (gavages) to 20 g mice. To determine the effect of XV454 on platelet aggregation, blood samples were collected by cardiac puncture 10 minutes after intravenous and 1-24 hrs after oral XV454, and platelet function was assessed by aggregometry, thrombelastography and the Platelet Function Analyzer (PFA100). The effect of XV454 on tumor cell-induced thrombocytopenia was determined 10 minutes after intravenous and 3 hrs after oral XV454 administration. Tumor cells (2 x 10(6)) were injected intravenously and 15 minutes after cell injection, platelet count was measured and compared to baseline (pre-injection) counts. To assess the effect on metastasis, XV454 was administered intravenous or orally 10 minutes and 3 hrs before tumor cell injection, respectively. Eighteen days later, surface lung tumor nodules were counted and the total lung tumor burden assessed. In a fourth group, in addition to the initial oral dose (before tumor cell injection), oral XV454 was given daily for the first week and three times in the second week. Administration of XV454 (5 mg/kg) completely inhibited platelet aggregation and this effect persisted for at least 24 hrs after oral delivery. Both intravenous and oral XV454 significantly inhibited tumor cell-induced thrombocytopenia (P < 0.01), the number of surface lung tumor nodules (80-85%; P

  5. Uterine cervix cancer treatment in IIB, IIIA and IIIB stages with external radiotherapy versus external radiotherapy and scintiscanning of low dose. ION SOLCA. Years 1998-2000

    International Nuclear Information System (INIS)

    Sanchez, Doris; Falquez, Roberto

    2002-01-01

    We realized study of retrospective accomplished in course of years 1998-2000, reviewing clinical charts of statistical department of ION SOLCA. We reviewed 544 cases in 1998, 603 patients in 1999, and 630 cases in 2000. In the radiotherapy service, 133 patients received treatment with external radiotherapy between February 1998 to February 1999 in IIB, IIIA, IIIB stages and only 80 patients were treated with external radiotherapy and scintiscanning of low dose rate in the same stages between March 1999 to March 2000. (The author)

  6. Alloimmunization against Iy, a low-frequency antigen on platelet glycoprotein Ib/IX as a cause of severe neonatal alloimmune thrombocytopenic purpura.

    Science.gov (United States)

    Kiefel, V; Vicariot, M; Giovangrandi, Y; Kroll, H; Böhringer, M; Greinacher, A; Breitfeld, C; Santoso, S; Mueller-Eckhardt, C

    1995-01-01

    Neonatal alloimmune thrombocytopenia (NAIT) is usually induced by platelet-specific antibodies against HPA-1a (Zwa) or HPA-5b (Bra). Recently, low-frequency alloantigens on the platelet glycoprotein (GP) IIb/IIIa complex have been discovered as a cause for NAIT. In this report, a new low-frequency platelet-specific alloantigen, Iy, is described which induced severe NAIT. The corresponding antigen was detected in 1/249 unrelated German blood donors. Antibody binding assays with trypsin-digested platelets (ELISA, immunoprecipitation with biotin-labelled platelets) indicate that the antigen is not localized on the glycocalicin moiety of GP Ib alpha, but may be situated on the remnant moiety of GP Ib alpha, GP IX or GPIb beta. Apparently, Iy is not related to the HPA-2 (Ko) antigen system.

  7. Impact of time to treatment on the effects of bivalirudin vs. glycoprotein IIb/IIIa inhibitors and heparin in patients undergoing primary percutaneous coronary intervention

    DEFF Research Database (Denmark)

    Schoos, Mikkel; De Luca, Giuseppe; Dangas, George D

    2016-01-01

    sought to examine whether these effects are dependent on time to treatment. METHODS AND RESULTS: The interaction between anticoagulation regimen and symptom onset to first balloon inflation time (SBT) on the 30-day and three-year rates of major adverse cardiac events (MACE) was examined in 3...

  8. The Optimal Route of Administration of the Glycoprotein IIb/IIIa Receptor Antagonist Abciximab During Percutaneous Coronary Intervention; Intravenous Versus Intracoronary

    DEFF Research Database (Denmark)

    Iversen, Allan; Galatius, Søren; Jensen, Jan S

    2008-01-01

    -randomised and retrospective studies or studies with short follow-up. No definite conclusion can be made based on these studies.In this review we present the current knowledge published about the intracoronary administration of Abciximab including the mechanisms behind the potential beneficial effects, and the safety...... and thrombus formation, but other mechanisms, such as suppression of the inflammatory pathways, have also been proposed to contribute to the benefits of Abciximab.The optimal route of administration, i.e. intravenous versus intracoronary, of the first dose has been questioned, but only tested in small, non...

  9. Dimers of beta 2-glycoprotein I mimic the in vitro effects of beta 2-glycoprotein I-anti-beta 2-glycoprotein I antibody complexes

    NARCIS (Netherlands)

    Lutters, B. C.; Meijers, J. C.; Derksen, R. H.; Arnout, J.; de Groot, P. G.

    2001-01-01

    Anti-beta(2)-glycoprotein I antibodies are thought to cause lupus anticoagulant activity by forming bivalent complexes with beta(2)-glycoprotein I (beta(2)GPI). To test this hypothesis, chimeric fusion proteins were constructed of the dimerization domain (apple 4) of factor XI and beta(2)GPI. Both a

  10. ST-segment resolution with bivalirudin versus heparin and routine glycoprotein IIb/IIIa inhibitors started in the ambulance in ST-segment elevation myocardial infarction patients transported for primary percutaneous coronary intervention

    DEFF Research Database (Denmark)

    Van't Hof, Arnoud; Giannini, Francesco; Ten Berg, Jurrien

    2017-01-01

    resolution either before or after the index procedure. CONCLUSIONS: Pre-hospital treatment with bivalirudin is non-inferior to pre-hospital heparin + GPI with regard to residual ST-segment deviation or ST-segment resolution, reflecting comparable myocardial reperfusion with the two strategies.......BACKGROUND: Myocardial reperfusion after primary percutaneous coronary intervention (PCI) can be assessed by the extent of post-procedural ST-segment resolution. The European Ambulance Acute Coronary Syndrome Angiography (EUROMAX) trial compared pre-hospital bivalirudin and pre-hospital heparin...... or enoxaparin with or without GPIIb/IIIa inhibitors (GPIs) in primary PCI. This nested substudy was performed in centres routinely using pre-hospital GPI in order to compare the impact of randomized treatments on ST-resolution after primary PCI. METHODS: Residual cumulative ST-segment deviation on the single...

  11. Dystrophin-glycoprotein complex sequesters Yap to inhibit cardiomyocyte proliferation.

    Science.gov (United States)

    Morikawa, Yuka; Heallen, Todd; Leach, John; Xiao, Yang; Martin, James F

    2017-07-13

    The regenerative capacity of the adult mammalian heart is limited, because of the reduced ability of cardiomyocytes to progress through mitosis. Endogenous cardiomyocytes have regenerative capacity at birth but this capacity is lost postnatally, with subsequent organ growth occurring through cardiomyocyte hypertrophy. The Hippo pathway, a conserved kinase cascade, inhibits cardiomyocyte proliferation in the developing heart to control heart size and prevents regeneration in the adult heart. The dystrophin-glycoprotein complex (DGC), a multicomponent transmembrane complex linking the actin cytoskeleton to extracellular matrix, is essential for cardiomyocyte homeostasis. DGC deficiency in humans results in muscular dystrophy, including the lethal Duchenne muscular dystrophy. Here we show that the DGC component dystroglycan 1 (Dag1) directly binds to the Hippo pathway effector Yap to inhibit cardiomyocyte proliferation in mice. The Yap-Dag1 interaction was enhanced by Hippo-induced Yap phosphorylation, revealing a connection between Hippo pathway function and the DGC. After injury, Hippo-deficient postnatal mouse hearts maintained organ size control by repairing the defect with correct dimensions, whereas postnatal hearts deficient in both Hippo and the DGC showed cardiomyocyte overproliferation at the injury site. In the hearts of mature Mdx mice (which have a point mutation in Dmd)-a model of Duchenne muscular dystrophy-Hippo deficiency protected against overload-induced heart failure.

  12. Dystrobrevin increases dystrophin's binding to the dystrophin-glycoprotein complex and provides protection during cardiac stress.

    Science.gov (United States)

    Strakova, Jana; Dean, Jon D; Sharpe, Katharine M; Meyers, Tatyana A; Odom, Guy L; Townsend, DeWayne

    2014-11-01

    Duchenne muscular dystrophy is a fatal progressive disease of both cardiac and skeletal muscle resulting from the mutations in the DMD gene and loss of the protein dystrophin. Alpha-dystrobrevin (α-DB) tightly associates with dystrophin but the significance of this interaction within cardiac myocytes is poorly understood. In the current study, the functional role of α-DB in cardiomyocytes and its implications for dystrophin function are examined. Cardiac stress testing demonstrated significant heart disease in α-DB null (adbn(-/-)) mice, which displayed mortality and lesion sizes that were equivalent to those seen in dystrophin-deficient mdx mice. Despite normal expression and subcellular localization of dystrophin in the adbn(-/-) heart, there is a significant decrease in the strength of dystrophin's interaction with the membrane-bound dystrophin-associated glycoprotein complex (DGC). A similar weakening of the dystrophin-membrane interface was observed in mice lacking the sarcoglycan complex. Cardiomyocytes from adbn(-/-) mice were smaller and responded less to adrenergic receptor induced hypertrophy. The basal decrease in size could not be attributed to aberrant Akt activation. In addition, the organization of the microtubule network was significantly altered in adbn(-/-) cardiac myocytes, while the total expression of tubulin was unchanged in adbn(-/-) hearts. These studies demonstrate that α-DB is a multifunctional protein that increases dystrophin's binding to the dystrophin-glycoprotein complex, and is critical for the full functionality of dystrophin. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. The ‘MAP strategy’ (Maximum aspiration of atherothrombus and adjunctive glycoprotein IIb/IIIa inhibitor utilization combined with prolonged inflation of balloon/stent for preventing no-reflow in patients with ST-segment elevation myocardial infarction undergoing percutaneous coronary intervention: A retrospective analysis of seventy-one cases

    Directory of Open Access Journals (Sweden)

    Anil Potdar

    2015-12-01

    Full Text Available ‘No-reflow’ phenomenon is a common occurrence in percutaneous coronary intervention (PCI. A three-component ‘MAP strategy’ was designed to prevent no-reflow by addressing both intralesional and intraluminal thrombus in patients with ST-segment elevation myocardial infarction (STEMI. In this analysis, we observed Thrombolysis In Myocardial Infarction (TIMI flow grade 3 or 2 in all patients, with no incidence of no-reflow. Myocardial blush grade (MBG 3 or 2 was observed in most (87.32% patients. Left ventricular ejection fraction (LVEF was improved, without any incidence of death up to 9-month follow-up. All patients safely tolerated the strategy-driven prolonged, 35-s inflation of the balloon/stent.

  14. Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1

    NARCIS (Netherlands)

    Sanders, Rogier W.; Vesanen, Mika; Schuelke, Norbert; Master, Aditi; Schiffner, Linnea; Kalyanaraman, Roopa; Paluch, Maciej; Berkhout, Ben; Maddon, Paul J.; Olson, William C.; Lu, Min; Moore, John P.

    2002-01-01

    The envelope glycoprotein (Env) complex of human immunodeficiency virus type I has evolved a structure that is minimally immunogenic while retaining its natural function of receptor-mediated virus-cell fusion. The Env complex is trimeric; its six individual subunits (three gp120 and three gp41

  15. The Dystrophin-Glycoprotein Complex in the Prevention of Muscle Damage

    Directory of Open Access Journals (Sweden)

    Jessica D. Gumerson

    2011-01-01

    Full Text Available Muscular dystrophies are genetically diverse but share common phenotypic features of muscle weakness, degeneration, and progressive decline in muscle function. Previous work has focused on understanding how disruptions in the dystrophin-glycoprotein complex result in muscular dystrophy, supporting a hypothesis that the muscle sarcolemma is fragile and susceptible to contraction-induced injury in multiple forms of dystrophy. Although benign in healthy muscle, contractions in dystrophic muscle may contribute to a higher degree of muscle damage which eventually overwhelms muscle regeneration capacity. While increased susceptibility of muscle to mechanical injury is thought to be an important contributor to disease pathology, it is becoming clear that not all DGC-associated diseases share this supposed hallmark feature. This paper outlines experimental support for a function of the DGC in preventing muscle damage and examines the evidence that supports novel functions for this complex in muscle that when impaired, may contribute to the pathogenesis of muscular dystrophy.

  16. Calcium-binding proteins from human platelets

    International Nuclear Information System (INIS)

    Gogstad, G.O.; Krutnes, M.B.; Solum, N.O.

    1983-01-01

    Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45 Ca 2 + and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45 Ca 2 + . These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4 Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45 Ca 2 + prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was wekly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelt-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIB-IIIa precipitate also became apparent. No increased incorporation of calcium occured in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45 Ca 2 + . (orig.)

  17. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

    OpenAIRE

    Kwong, Peter D.; Wyatt, Richard; Robinson, James; Sweet, Raymond W.; Sodroski, Joseph; Hendrickson, Wayne A.

    1998-01-01

    The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gpl20 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 Å resolution of an HIV-1 gp120 core complexed with a two-domain fra...

  18. Dystrophin-glycoprotein complex regulates muscle nitric oxide production through mechanoregulation of AMPK signaling.

    Science.gov (United States)

    Garbincius, Joanne F; Michele, Daniel E

    2015-11-03

    Patients deficient in dystrophin, a protein that links the cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex (DGC), exhibit muscular dystrophy, cardiomyopathy, and impaired muscle nitric oxide (NO) production. We used live-cell NO imaging and in vitro cyclic stretch of isolated adult mouse cardiomyocytes as a model system to investigate if and how the DGC directly regulates the mechanical activation of muscle NO signaling. Acute activation of NO synthesis by mechanical stretch was impaired in dystrophin-deficient mdx cardiomyocytes, accompanied by loss of stretch-induced neuronal NO synthase (nNOS) S1412 phosphorylation. Intriguingly, stretch induced the acute activation of AMP-activated protein kinase (AMPK) in normal cardiomyocytes but not in mdx cardiomyocytes, and specific inhibition of AMPK was sufficient to attenuate mechanoactivation of NO production. Therefore, we tested whether direct pharmacologic activation of AMPK could bypass defective mechanical signaling to restore nNOS activity in dystrophin-deficient cardiomyocytes. Indeed, activation of AMPK with 5-aminoimidazole-4-carboxamide riboside or salicylate increased nNOS S1412 phosphorylation and was sufficient to enhance NO production in mdx cardiomyocytes. We conclude that the DGC promotes the mechanical activation of cardiac nNOS by acting as a mechanosensor to regulate AMPK activity, and that pharmacologic AMPK activation may be a suitable therapeutic strategy for restoring nNOS activity in dystrophin-deficient hearts and muscle.

  19. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody.

    Science.gov (United States)

    Kwong, P D; Wyatt, R; Robinson, J; Sweet, R W; Sodroski, J; Hendrickson, W A

    1998-06-18

    The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gp120 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 A resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.

  20. The Complexity of Antibody Responses Elicited against the Respiratory Syncytial Virus Glycoproteins in Hospitalized Children Younger than 2 Years

    Directory of Open Access Journals (Sweden)

    Alfonsina Trento

    2017-11-01

    Full Text Available The influence of age and maternal antibodies on the antibody responses to human respiratory syncytial virus (hRSV glycoproteins in very young children has been a matter of controversy. Both, immaturity of the immune system at very early age and suppression of the host immune response by high level of maternal antibodies have been claimed to limit the host antibody response to virus infection and to jeopardize the use of hRSV vaccines under development in that age group. Hence, the antibody responses to the two major hRSV glycoproteins (F and G were evaluated in children younger than 2 years, hospitalized with laboratory confirmed hRSV bronchiolitis. A strong negative correlation was found between the titre of circulating ELISA antibodies directed against either prefusion or postfusion F in the acute phase, but not age, and their fold change at convalescence. These changes correlated also with the level of circulating neutralizing antibodies in sera. As reported in adults, most neutralizing antibodies in a subset of tested sera could not be depleted with postfusion F, suggesting that they were mostly directed against prefusion-specific epitopes. In contrast, a weak negative association was found for group-specific anti-G antibodies in the acute phase and their fold change at convalescence only after correcting for the antigenic group of the infecting virus. In addition, large discrepancies were observed in some individuals between the antibody responses specific for F and G glycoproteins. These results illustrate the complexity of the anti-hRSV antibody responses in children experiencing a primary severe infection and the influence of preexisting maternal antibodies on the host response, factors that should influence hRSV serological studies as well as vaccine development.

  1. The Structure of Herpesvirus Fusion Glycoprotein B-Bilayer Complex Reveals the Protein-Membrane and Lateral Protein-Protein Interaction

    NARCIS (Netherlands)

    Maurer, Ulrike E.; Zeev-Ben-Mordehai, Tzviya; Pandurangan, Arun Prasad; Cairns, Tina M.; Hannah, Brian P.; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.; Topf, Maya; Huiskonen, Juha T.; Gruenewald, Kay

    2013-01-01

    Glycoprotein B (gB) is a key component of the complex herpesvirus fusion machinery. We studied membrane interaction of two gB ectodomain forms and present an electron cryotomography structure of the gB-bilayer complex. The two forms differed in presence or absence of the membrane proximal region

  2. Albumin is synthesized in epididymis and aggregates in a high molecular mass glycoprotein complex involved in sperm-egg fertilization.

    Directory of Open Access Journals (Sweden)

    Kélen Fabíola Arroteia

    Full Text Available The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.

  3. Albumin is synthesized in epididymis and aggregates in a high molecular mass glycoprotein complex involved in sperm-egg fertilization.

    Science.gov (United States)

    Arroteia, Kélen Fabíola; Barbieri, Mainara Ferreira; Souza, Gustavo Henrique Martins Ferreira; Tanaka, Hiromitsu; Eberlin, Marcos Nogueira; Hyslop, Stephen; Alvares, Lúcia Elvira; Pereira, Luís Antonio Violin Dias

    2014-01-01

    The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.

  4. New insights into the Hendra virus attachment and entry process from structures of the virus G glycoprotein and its complex with Ephrin-B2.

    Directory of Open Access Journals (Sweden)

    Kai Xu

    Full Text Available Hendra virus and Nipah virus, comprising the genus Henipavirus, are recently emerged, highly pathogenic and often lethal zoonotic agents against which there are no approved therapeutics. Two surface glycoproteins, the attachment (G and fusion (F, mediate host cell entry. The crystal structures of the Hendra G glycoprotein alone and in complex with the ephrin-B2 receptor reveal that henipavirus uses Tryptophan 122 on ephrin-B2/B3 as a "latch" to facilitate the G-receptor association. Structural-based mutagenesis of residues in the Hendra G glycoprotein at the receptor binding interface document their importance for viral attachments and entry, and suggest that the stability of the Hendra-G-ephrin attachment complex does not strongly correlate with the efficiency of viral entry. In addition, our data indicates that conformational rearrangements of the G glycoprotein head domain upon receptor binding may be the trigger leading to the activation of the viral F fusion glycoprotein during virus infection.

  5. Glycoprotein on cell surfaces

    International Nuclear Information System (INIS)

    Muramatsu, T.

    1975-01-01

    There are conjugated polysaccharides in cell membranes and outside of animal cells, and they play important role in the control of cell behavior. In this paper, the studies on the glycoprotein on cell surfaces are reported. It was found that the glycoprotein on cell surfaces have both N-glycoside type and O-glycoside type saccharic chains. Therefore it can be concluded that the basic structure of the saccharic chains in the glycoprotein on cell surfaces is similar to that of blood serum and body fluid. The main glycoprotein in the membranes of red blood corpuscles has been studied most in detail, and it also has both types of saccharic chains. The glycoprotein in liver cell membranes was found to have only the saccharic chains of acid type and to be in different pattern from that in endoplasmic reticula and nuclear membranes, which also has the saccharic chains of neutral type. The structure of the saccharic chains of H-2 antigen, i.e. the peculiar glycoprotein on the surfaces of lymph system cells, has been studied, and it is similar to the saccharic chains of glycoprotein in blood serum. The saccharic chain structures of H-2 antigen and TL antigen are different. TL, H-2 (D), Lna and H-2 (K) are the glycoprotein on cell surfaces, and are independent molecules. The analysis of the saccharic chain patterns on cell surfaces was carried out, and it was shown that the acid type saccharic chains were similar to those of ordinary glycoprotein, because the enzyme of pneumococci hydrolyzed most of the acid type saccharic chains. The change of the saccharic chain patterns of glycoprotein on cell surfaces owing to canceration and multiplication is complex matter. (Kako, I.)

  6. Role of Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast Cancer Cells

    National Research Council Canada - National Science Library

    Zolov, Sergey N

    2006-01-01

    ...: protein glycosylation and its sorting. For analysis of COG complex function we utilized RNA interference assay to knockdown COG3p subunit of COG complex in normal and breast cancer cells and other tumor cell lines...

  7. Bivalirudin Started during Emergency Transport for Primary PCI

    DEFF Research Database (Denmark)

    Steg, Philippe Gabriel; van 't Hof, Arnoud; Hamm, Christian W

    2013-01-01

    Bivalirudin, as compared with heparin and glycoprotein IIb/IIIa inhibitors, has been shown to reduce rates of bleeding and death in patients undergoing primary percutaneous coronary intervention (PCI). Whether these benefits persist in contemporary practice characterized by prehospital initiation...

  8. Heart Truth for Women: If You Have Heart Disease

    Science.gov (United States)

    ... when it contracts. C 2 Nuclear scan (or thallium stress test) can be used for three main ... channel blockers; nitroglycerin; glycoprotein IIb-IIIa; statins; and fish oil and other supplements high in omega-3 ...

  9. Host cell recognition by the henipaviruses: Crystal structures of the Nipah G attachment glycoprotein and its complex with ephrin-B3

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Kai; Rajashankar, Kanagalaghatta R.; Chan, Yee-Peng; Himanen, Juha P.; Broder, Christopher C.; Nikolov, Dimitar B. (USUHS); (Cornell); (MSKCC)

    2008-07-28

    Nipah virus (NiV) and Hendra virus are the type species of the highly pathogenic paramyxovirus genus Henipavirus, which can cause severe respiratory disease and fatal encephalitis infections in humans, with case fatality rates approaching 75%. NiV contains two envelope glycoproteins, the receptor-binding G glycoprotein (NiV-G) that facilitates attachment to host cells and the fusion (F) glycoprotein that mediates membrane merger. The henipavirus G glycoproteins lack both hemagglutinating and neuraminidase activities and, instead, engage the highly conserved ephrin-B2 and ephrin-B3 cell surface proteins as their entry receptors. Here, we report the crystal structures of the NiV-G both in its receptor-unbound state and in complex with ephrin-B3, providing, to our knowledge, the first view of a paramyxovirus attachment complex in which a cellular protein is used as the virus receptor. Complex formation generates an extensive protein-protein interface around a protruding ephrin loop, which is inserted in the central cavity of the NiV-G {beta}-propeller. Analysis of the structural data reveals the molecular basis for the highly specific interactions of the henipavirus G glycoproteins with only two members (ephrin-B2 and ephrin-B3) of the very large ephrin family and suggests how they mediate in a unique fashion both cell attachment and the initiation of membrane fusion during the virus infection processes. The structures further suggest that the NiV-G/ephrin interactions can be effectively targeted to disrupt viral entry and provide the foundation for structure-based antiviral drug design.

  10. A multigene family encodes the human cytomegalovirus glycoprotein complex gcII (gp47-52 complex)

    International Nuclear Information System (INIS)

    Gretch, D.R.; Stinski, M.F.; Kari, B.; Gehrz, R.C.

    1988-01-01

    The HXLF (HindIII-X left reading frame) gene family is a group of five genes that share one or two regions of homology and are arranged in tandem within the short unique component of the human cytomegalovirus genome. These genes were cloned into an SP6 expression vector in both the sense and antisense orientations. An abundant 1.62-kilobase (kb) bicistronic mRNA, predicted to originate from HXLF1 and HXLF2, was detected in the cytoplasm of infected human fibroblast cells by Northern (RNA) blot analysis. Less abundant RNAs of 1.0 and 0.8 kb, predicted to originate from the HXLF5 and HXLF2 genes, respectively, were also detected. Monocistronic, bicistronic, and polycistronic RNAs synthesized in vitro by using SP6 polymerase were translated in rabbit reticulocyte lysates with or without canine pancreatic microsomal membranes. The HXLF1 or the HXLF1 and HXLF2 translation products were detected when the above mRNAs were used. The HXLF3, HXLF4, and HXLF5 gene products were not detected by in vitro translation of the SP6-derived polycistronic mRNA. The amino acid composition of gp47-52 purified from virion envelopes has the highest similarity to the predicted amino acid composition of the HXLF1 plus HXLF2 open reading frames, but it is more similar to HXLF2 than to HXLF1. The Northern blot results imply that gp47-52 is synthesized predominantly from the abundant 1.62-kb bicistronic mRNA encoded by the HXLF1 and HXLF2 genes. However, the glycoprotein could also be synthesized by the monocistronic 0.8-kb mRNA encoded by the HXLF2 gene as well as by the mRNAs predicted from the other HXLF genes

  11. Efficacy and Safety of Platelet Glycoprotein Receptor Blockade in Aged and Comorbid Mice With Acute Experimental Stroke.

    Science.gov (United States)

    Kraft, Peter; Schuhmann, Michael K; Fluri, Felix; Lorenz, Kristina; Zernecke, Alma; Stoll, Guido; Nieswandt, Bernhard; Kleinschnitz, Christoph

    2015-12-01

    Despite the medical and socioeconomic effect of ischemic stroke and extensive preclinical research, treatment options for ischemic stroke are limited. We recently identified and characterized essential steps of thrombus formation in stroke and demonstrated that inhibition of the platelet glycoprotein (GP) receptors Ib and VI, but not IIb/IIIa, protects young and healthy mice from ischemic neurodegeneration. Whether these findings translate to the clinic remains unclear. Considering that the typical stroke patient is elderly with comorbidity, we aimed to analyze the efficacy and safety of novel preclinical antithrombotics in adult and comorbid mice with acute experimental stroke. We subjected adult, healthy, atherosclerotic (Ldlr(-/-)), diabetic (streptozotocin treated), and hypertensive (RenTgMK) mice to a 60-minute transient middle cerebral artery occlusion. Animals were pretreated with anti-GPVI antibodies or treated 1 hour after stroke induction with anti-GPIb or anti-GPIIb/IIIa antigen-binding fragments, respectively. Isotype treatment served as control. Twenty-four hours after transient middle cerebral artery occlusion, we visually assessed the intracerebral hemorrhage rate and measured infarct volumes (using 2,3,5-triphenyltetrazolium chloride-stained brain slices) and functional outcome (using Bederson and grip-test scores). GPIb and GPVI inhibition protected the mice from ischemic stroke without increasing bleeding complications. In contrast, GPIIb/IIIa inhibition was not protective but increased the intracerebral hemorrhage rate. Inhibition of early steps of thrombus formation protects adult and comorbid mice from ischemic stroke. The use of clinically meaningful mouse strains might improve the translation of preclinical stroke research to the clinic. © 2015 American Heart Association, Inc.

  12. A V740L mutation in glycoprotein IIb defines a novel epitope (War) associated with fetomaternal alloimmune thrombocytopenia.

    Science.gov (United States)

    Poles, Anthony; Woźniak, Marcin J; Walser, Piers; Ridgwell, Kay; Fitzgerald, Joan; Green, Ann; Gilmore, Ruth; Lucas, Geoff

    2013-09-01

    Most recently described human platelet antigens (HPAs) have been detected in cases of fetomaternal alloimmune thrombocytopenia (FMAIT) where the mother has been immunized against a low-frequency antigen that the fetus has inherited from the father. Low-frequency antigens are not represented in normal panel platelets (PLTs) and antibody detection and identification in such cases requires incubation of maternal serum with paternal PLTs and definition of the causative mutation. A suspected case of FMAIT was investigated for PLT-specific antibodies using a panel of both HPA-typed and paternal PLTs. HPA typing was performed by polymerase chain reaction with sequence-specific primers and further DNA analysis was performed using direct sequencing of the coding regions of the ITGA2B and ITGB3 genes. Maternal antibodies reactive only with paternal PLTs were localized to glycoprotein (GP)IIb/IIIa using the monoclonal antibody immobilization of PLT antibody assay. A single-nucleotide polymorphism was detected in Exon 23 of ITGA2B in the father and affected child, which predicted a V740L substitution in the mature protein. Recombinant V740L mutated GPIIb expressed in HEK293 cells was specifically recognized by maternal antibodies. The polymorphism was not detected either in the mother or in a cohort of 100 donors. The V740L polymorphism defines a new low-frequency antigen implicated in two cases of FMAIT in a single family. Low-frequency HPAs are clinically important and their elucidation requires both crossmatch studies and gene sequencing in cases where there is strong clinical evidence of FMAIT but initial laboratory investigations do not support the diagnosis. © 2013 American Association of Blood Banks.

  13. The potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex via alpha-syntrophin in glia.

    Science.gov (United States)

    Connors, Nathan C; Adams, Marvin E; Froehner, Stanley C; Kofuji, Paulo

    2004-07-02

    One of the major physiological roles of potassium channels in glial cells is to promote "potassium spatial buffering" in the central nervous system, a process necessary to maintain an optimal potassium concentration in the extracellular environment. This process requires the precise distribution of potassium channels accumulated at high density in discrete subdomains of glial cell membranes. To obtain a better understanding of how glial cells selectively target potassium channels to discrete membrane subdomains, we addressed the question of whether the glial inwardly rectifying potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex (DGC). Immunoprecipitation experiments revealed that Kir4.1 is associated with the DGC in mouse brain and cultured cortical astrocytes. In vitro immunoprecipitation and pull-down assays demonstrated that Kir4.1 can bind directly to alpha-syntrophin, requiring the presence of the last three amino acids of the channel (SNV), a consensus PDZ domain-binding motif. Furthermore, Kir4.1 failed to associate with the DGC in brains from alpha-syntrophin knockout mice. These results suggest that Kir4.1 is localized in glial cells by its association with the DGC through a PDZ domain-mediated interaction with alpha-syntrophin and suggest an important role for the DGC in central nervous system physiology.

  14. Age-related dystrophin-glycoprotein complex structure and function in the rat extensor digitorum longus and soleus muscle.

    Science.gov (United States)

    Rice, Kevin M; Preston, Deborah L; Neff, David; Norton, Michael; Blough, Eric R

    2006-11-01

    This study tested the hypothesis that age-related changes in the dystrophin-glycoprotein complex (DGC) may precede age-associated alterations in muscle morphology and function. Compared to those in adult (6 month) rats, extensor digitorum longus (EDL) and soleus muscle mass was decreased in old (30 month) and very old (36 month) Fischer 344/NNiaHSD x Brown Norway/BiNia rats. The amount of dystrophin, beta-dystroglycan, and alpha-sarcoglycan increased with aging in the EDL and decreased with aging in the soleus. alpha-Dystroglycan levels were increased with aging in both muscles and displayed evidence of altered glycosylation. Immunostaining for the presence of antibody infiltration and dystrophin following increased muscle stretch suggested that the aging in the soleus was characterized by diminished membrane integrity. Together, these data suggest that aging is associated with alterations in EDL and soleus DGC protein content and localization. These results may implicate the DGC as playing a role in age-associated skeletal muscle remodeling.

  15. Myelin-oligodendrocyte glycoprotein is a member of a subset of the immunoglobulin superfamily encoded within the major histocompatibility complex

    Energy Technology Data Exchange (ETDEWEB)

    Pham-Dinh, D.; Dautigny, A. (Institut des Neurosciences, Paris (France)); Mattei, M.G.; Roeckel, N. (Institut National de la Sante et de la Recherche Medicale Unite, Marseille (France)); Nussbaum, J.H.; Roussel, G. (Centre National de la Recherche Scientifique Unite, Strasbourg (France)); Pontarotti, P. (Centre Natinal de la Recherche Scientifique Unite, Toulouse (France)); Mather, I.H. (Univ. of Maryland, College Park, MD (United States)); Artzt, K. (Univ. of Texas, Austin, TX (United States)); Lindahl, K.F. (Univ. of Texas Southwestern Medical Center, Dallas, TX (United States))

    1993-09-01

    Myelin/oligodendrocyte glycoprotein (MOG) is found on the surface of myelinating oligodendrocytes and external lamellae of myelin sheaths in the central nervous system, and it is target antigen in experimental autoimmune encephalomyelitis and multiple sclerosis. The authors have isolated bovine, mouse, and rat MOG cDNA clones and shown that the developmental pattern of MOG expression in the rat central nervous system coincides with the late stages of myelination. The amino-terminal, extracellular domain of MOG has characteristics of an immunoglobulin variable domain and is 46% and 41% identical with the amino terminus of bovine butyrophilin (expressed in the lactating mammary gland) and B-G antigens of the chicken major histocompatibility complex (MHC), respectively; these proteins thus form a subset of the immunoglobulin superfamily. The homology between MOG and B-G extends beyond their structure and genetic mapping to their ability to induce strong antibody responses and has implications for the role of MOG in pathological, autoimmune conditions. The authors colocalized the MOG and BT genes to the human MHC on chromosome 6p21.3-p22. The mouse MOG gene was mapped to the homologous band C of chromosome 17, within the M region of the mouse MHC. 38 refs., 6 figs.

  16. The PSA-2 glycoprotein complex of Leishmania major is a glycosylphosphatidylinositol-linked promastigote surface antigen.

    Science.gov (United States)

    Murray, P J; Spithill, T W; Handman, E

    1989-12-15

    Polyclonal rabbit antiserum to the Triton X-114 phase material of Leishmania major, which comprises the surface and internal integral membrane proteins of the parasite, was used to screen a lambda gt11 genomic expression library. A recombinant clone producing a Mr 123,000 beta-galactosidase fusion protein was isolated. Antibodies affinity-purified on this fusion protein recognized a complex of three surface-oriented proteins of promastigotes of L. major of Mr 94,000, 90,000, and 80,000 that we have termed the promastigote surface Ag 2 (PSA-2) complex. The DNA sequence of the insert in this clone predicted the 3' end of an open reading frame encoding a hydrophobic C-terminus. The inferred C-terminal sequence was suggestive of a glycosylphosphatidyl-inositol membrane anchoring mechanism. Phosphatidylinositol-specific phospholipase C treatment of the native PSA-2 proteins caused a shift in their electrophoretic mobility with an apparent reduction in the molecular weight of the PSA-2 complex. After phospholipase C treatment these proteins also displayed the cryptic cross-reacting determinant recognized by antibodies to the Trypanosoma brucei variant surface Ag. Moreover, PSA-2, which previously partitioned in the detergent phase after Triton X-114 phase separation, became water-soluble after phospholipase C treatment. Immunoprecipitation of the PSA-2 proteins with sera directed to lectin-binding proteins indicated that these polypeptides may be differentially glycosylated. Finally, these PSA-2 proteins were recognized by sera from some patients with cutaneous leishmaniasis.

  17. Microfilament association of ASGP-2, the concanavalin A-binding glycoprotein of the cell-surface sialomucin complex of 13762 rat mammary ascites tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Vanderpuye, L.A.; Carraway, C.A.C.; Carraway, K.L. (Univ. of Miami School of Medicine, FL (USA))

    1988-10-01

    Microfilament-associated proteins and membrane-microfilament interactions are being investigated in microvilli isolated from 13762 rat mammary ascites tumor cells. Phalloidin shift analyses on velocity sedimentation gradients of Triton X-100 extracts of ({sup 3}H)-glucosamine-labeled microvilli identified a 120-kDa cell-surface glycoprotein associated with the microvillar microfilament core. The identification was verified by concanavalin A (Con A) blots of one- and two-dimensional (2D) electrophoresis gels of sedimented microfilament cores. By 2D-electrophoresis and lectin analyses the 120-kDa protein appeared to be a fraction of ASGP-2, the major Con A-binding glycoprotein of the sialomucin complex of the 13762 cells. This identity was confirmed by immunoblot analyses using immunoblot-purified anti-ASGP-2 from anti-membrane serum prepared against microvillar membranes. Proteolysis of the microvilli with subtilisin or trypsin resulted in an increase in the amount of ASGP-2 associated with the microfilament cores. Proteolysis of isolated microvillar membranes, which contain actin but not microfilaments, also increased the association of ASGP-2 with a Triton-insoluble, actin-containing membrane fraction. Since the Triton-insoluble membrane residue is enriched in actin-containing transmembrane complex, which contains a different glycoprotein, the authors suggest that the ASGP-2 is binding indirectly via this complex to the microfilament core in the intact microvilli.

  18. Complex interplay between the P-glycoprotein multidrug efflux pump and the membrane: its role in modulating protein function

    Directory of Open Access Journals (Sweden)

    Frances Jane Sharom

    2014-03-01

    Full Text Available Multidrug resistance in cancer is linked to expression of the P-glycoprotein multidrug transporter (Pgp, ABCB1, which exports many structurally diverse compounds from cells. Substrates first partition into the bilayer and then interact with a large flexible binding pocket within the transporter’s transmembrane regions. Pgp has been described as a hydrophobic vacuum cleaner or an outwardly-directed drug/lipid flippase. Recent X-ray crystal structures have shed some light on the nature of the drug-binding pocket and suggested routes by which substrates can enter it from the membrane. Detergents have profound effects on Pgp function, and several appear to be substrates. Biochemical and biophysical studies in vitro, some using purified reconstituted protein, have explored the effects of the membrane environment. They have demonstrated that Pgp is involved in a complex relationship with its lipid environment, which modulates the behaviour of its substrates, as well as various functions of the protein, including ATP hydrolysis, drug binding and drug transport. Membrane lipid composition and fluidity, phospholipid headgroup and acyl chain length all influence Pgp function. Recent studies focusing on thermodynamics and kinetics have revealed some important principles governing Pgp-lipid and substrate-lipid interactions, and how these affect drug binding and transport. In some cells, Pgp is associated with cholesterol-rich microdomains which may modulate its functions. The relationship between Pgp and cholesterol remains an open question; however it clearly affects several aspects of its function in addition to substrate-membrane partitioning. The action of Pgp modulators appears to depend on their membrane permeability, and membrane fluidizers and surfactants reverse drug resistance, likely via an indirect mechanism. A detailed understanding of how the membrane affects Pgp substrates and Pgp’s catalytic cycle may lead to new strategies to combat

  19. Alloantibody against new platelet alloantigen (Lap(a)) on glycoprotein IIb is responsible for a case of fetal and neonatal alloimmune thrombocytopenia.

    Science.gov (United States)

    Wihadmadyatami, Hevi; Heidinger, Kathrin; Röder, Lida; Werth, Silke; Giptner, Astrid; Hackstein, Holger; Knorr, Martin; Bein, Gregor; Sachs, Ulrich J; Santoso, Sentot

    2015-12-01

    Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by the destruction of platelets (PLTs) in the fetus or newborn by maternal PLT antibodies that crossed the placenta during pregnancy. In this study, we aim to elucidate the properties of a new PLT alloantigen (Lap(a)) that is associated with a severe case of FNAIT. Analysis of maternal serum with phenotyped PLTs by monoclonal antibody-specific immobilization of platelet antigens showed positive reaction against PLT glycoprotein (GP)IIb/IIIa and HLA Class I expressed on paternal PLTs. In contrast to GPIIIa-reactive anti-HPA-1a, anti-Lap(a) alloantibodies precipitated predominantly GPIIb. Indeed, a point mutation G>C at Position 2511 located in Exon 25 of the ITGA2B gene was found in Lap(a)-positive donors. This mutation causes an amino exchange Gln>His at Position 806 located in the calf-2 domain of GPIIb. Lap(a)-positive individuals were not found in 300 random blood donors. Our expression study showed that anti-Lap(a) alloantibodies reacted with stable transfected HEK293 cells expressing the mutated GPIIb isoform (His806). CHO cells carrying this isoform, however, failed to react with anti-Lap(a) alloantibodies, indicating that Lap(a) epitopes depend on the Gln806 His mutation and the carbohydrate composition of the GPIIb. This mutation did not hamper the binding of anti-HPA-3a, which recognizes a point mutation (Ile843 Ser) located in calf-2 domain. Finally, we found that Lap(a) and some HPA-3a epitopes are sensitive to O-glycanase. This study not only underlines the relevance of rare HPAs on the pathomechanism of FNAIT, but also helps to understand the pitfalls of serologic assays to detect anti-GPIIb alloantibodies. © 2015 AABB.

  20. Simultaneous Pathoproteomic Evaluation of the Dystrophin-Glycoprotein Complex and Secondary Changes in the mdx-4cv Mouse Model of Duchenne Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Sandra Murphy

    2015-06-01

    Full Text Available In skeletal muscle, the dystrophin-glycoprotein complex forms a membrane-associated assembly of relatively low abundance, making its detailed proteomic characterization in normal versus dystrophic tissues technically challenging. To overcome this analytical problem, we have enriched the muscle membrane fraction by a minimal differential centrifugation step followed by the comprehensive label-free mass spectrometric analysis of microsomal membrane preparations. This organelle proteomic approach successfully identified dystrophin and its binding partners in normal versus dystrophic hind limb muscles. The introduction of a simple pre-fractionation step enabled the simultaneous proteomic comparison of the reduction in the dystrophin-glycoprotein complex and secondary changes in the mdx-4cv mouse model of dystrophinopathy in a single analytical run. The proteomic screening of the microsomal fraction from dystrophic hind limb muscle identified the full-length dystrophin isoform Dp427 as the most drastically reduced protein in dystrophinopathy, demonstrating the remarkable analytical power of comparative muscle proteomics. Secondary pathoproteomic expression patterns were established for 281 proteins, including dystrophin-associated proteins and components involved in metabolism, signalling, contraction, ion-regulation, protein folding, the extracellular matrix and the cytoskeleton. Key findings were verified by immunoblotting. Increased levels of the sarcolemmal Na+/K+-ATPase in dystrophic leg muscles were also confirmed by immunofluorescence microscopy. Thus, the reduction of sample complexity in organelle-focused proteomics can be advantageous for the profiling of supramolecular protein complexes in highly intricate systems, such as skeletal muscle tissue.

  1. The M/GP(5 glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner.

    Directory of Open Access Journals (Sweden)

    Wander Van Breedam

    2010-01-01

    Full Text Available The porcine reproductive and respiratory syndrome virus (PRRSV is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP(3, GP(4 and GP(5 envelope glycoproteins, only the M/GP(5 glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP(5. These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines.

  2. The complexity of roles of P-glycoprotein in refractory epilepsy: Pharmacoresistance, epileptogenesis, SUDEP and relapsing marker after surgical treatment

    Directory of Open Access Journals (Sweden)

    Alberto Lazarowski

    2015-07-01

    Full Text Available As described initially from clinical and experimental studies, P-glycoprotein (P-gp plays a central role in the pharmacoresistance of epilepsy, acting by efflux of AEDs mainly at blood brain barrier (BBB level. However, repetitive seizures can produce both brain and heart P-gp overexpression. Because P-gp activity induces membrane depolarization, its neuronal expression could be acting in the intrinsic mechanism of epileptogenesis, and its heart expression, can be a high risk factor of death, after severe-continuo convulsive stresses as in  fatal status epilepticus or in SUDEP. Additionally, because P-gp is also a stem cell marker, we suggests that its constitutive overexpression in dysplastic neurons from brain epileptogenic areas observed in patients with refractory epilepsies, should be addressed as a risk factor of seizures relapse after surgical treatment. Here we discuss these concepts, based on our own clinical and experimental experiences, and reviewing the current literature on these subjects.

  3. Antibodies Against β2-Glycoprotein I Complexed With an Oxidised Lipoprotein Relate to Intima Thickening of Carotid Arteries in Primary Antiphospholipid Syndrome

    Directory of Open Access Journals (Sweden)

    P. R. J. Ames

    2006-01-01

    Full Text Available To explore whether antibodies against β2-glycoprotein I (β2GPI complexed to 7-ketocholesteryl-9-carboxynonanoate (oxLig-1 and to oxidised low-density lipoproteins (oxLDL relate to paraoxonase activity (PONa and/or intima media thickness (IMT of carotid arteries in primary antiphospholipid syndrome (PAPS. As many as 29 thrombotic patients with PAPS, 10 subjects with idiopathic antiphospholipid antibodies (aPL without thrombosis, 17 thrombotic patients with inherited thrombophilia and 23 healthy controls were investigated. The following were measured in all participants: β2GPI−oxLDL complexes, IgG anti-β2GPI−oxLig-1, IgG anti-β2GPI−oxLDL antibodies (ELISA, PONa, (para-nitrophenol method, IMT of common carotid (CC artery, carotid bifurcation (B, internal carotid (IC by high resolution sonography. β2GPI−oxLDL complex was highest in the control group (p < 0.01, whereas, IgG anti-β2GPI−oxLig1 and IgG anti-β2GPI−oxLDL were highest in PAPS (p < 0.0001. In healthy controls, β2GPI−oxLDL complexes positively correlated to IMT of the IC (p = 0.007 and negatively to PONa after correction for age (p < 0.03. PONa inversely correlated with age (p = 0.008. In PAPS, IgG anti-2GPI−oxLig-1 independently predicted PONa (p = 0.02 and IMT of B (p = 0.003, CC, (p = 0.03 and of IC (p = 0.04. In PAPS, PONa inversely correlated to the IMT of B, CC and IC (p = 0.01, 0.02 and 0.003, respectively. IgG anti-2GPI−oxLig-1 may be involved in PAPS related atherogenesis via decreased PON activity.

  4. Epistastic Interactions within the Junín Virus Envelope Glycoprotein Complex Provide an Evolutionary Barrier to Reversion in the Live-Attenuated Candid#1 Vaccine.

    Science.gov (United States)

    York, Joanne; Nunberg, Jack H

    2018-01-01

    The Candid#1 strain of Junín virus was developed using a conventional attenuation strategy of serial passage in nonhost animals and cultured cells. The live-attenuated Candid#1 vaccine is used in Argentina to protect at-risk individuals against Argentine hemorrhagic fever, but it has not been licensed in the United States. Recent studies have revealed that Candid#1 attenuation is entirely dependent on a phenylalanine-to-isoleucine substitution at position 427 in the fusion subunit (GP2) of the viral envelope glycoprotein complex (GPC), thereby raising concerns regarding the potential for reversion to virulence. In this study, we report the identification and characterization of an intragenic epistatic interaction between the attenuating F427I mutation in GP2 and a lysine-to-serine mutation at position 33 in the stable signal peptide (SSP) subunit of GPC, and we demonstrate the utility of this interaction in creating an evolutionary barrier against reversion to the pathogenic genotype. In the presence of the wild-type F427 residue, the K33S mutation abrogates the ability of ectopically expressed GPC to mediate membrane fusion at endosomal pH. This defect is rescued by the attenuating F427I mutation. We show that the recombinant Candid#1 (rCan) virus bearing K33S GPC is viable and retains its attenuated genotype under cell culture conditions that readily select for reversion in the parental rCan virus. If back-mutation to F427 offers an accessible pathway to increase fitness in rCan, reversion in K33S-GPC rCan is likely to be lethal. The epistatic interaction between K33S and F427I thus may minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine. IMPORTANCE The live-attenuated Candid#1 vaccine strain of Junín virus is used to protect against Argentine hemorrhagic fever. Recent findings that a single missense mutation in the viral envelope glycoprotein complex (GPC) is responsible for attenuation raise the prospect of facile

  5. Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI

    International Nuclear Information System (INIS)

    Johnson, D.C.; Ligas, M.W.; Frame, M.C.; Cross, A.M.; Stow, N.D.

    1988-01-01

    Evidence was recently presented that herpes simplex virus type 1 (HSV-1) immunoglobulin G (IgG) Fc receptors are composed of a complex containing a previously described glycoprotein, gE, and a novel virus-induced polypeptide, provisionally named g70. Using a monoclonal antibody designated 3104, which recognizes g70, in conjunction with antipeptide sera and virus mutants unable to express g70 or gE, the authors have mapped the gene encoding g70 to the US7 open reading frame of HSV-1 adjacent to the gE gene. Therefore, g70 appears to be identical to a recently described polypeptide which was named gI. Under mildly denaturing conditions, monoclonal antibody 3104 precipitated both gI and gE from extracts of HSV-1-infected cells. In addition, rabbit IgG precipitated the gE-gI complex from extracts of cells transfected with a fragment of HSV-1 DNA containing the gI, gE, and US9 genes. Cells infected with mutant viruses which were unable to express gE or gI did not bind radiolabeled IgG; however, cells coinfected with two viruses, one unable to express gE and the other unable to express gI, bound levels of IgG approaching those observed with wild-type viruses. These results further support the hypothesis that gE and gI form a complex which binds IgG by the Fc domain and that neither polypeptide alone can bind IgG

  6. HCMV Infection of Human Trophoblast Progenitor Cells of the Placenta Is Neutralized by a Human Monoclonal Antibody to Glycoprotein B and Not by Antibodies to the Pentamer Complex

    Directory of Open Access Journals (Sweden)

    Martin Zydek

    2014-03-01

    Full Text Available Human cytomegalovirus (HCMV is the major viral cause of congenital infection and birth defects. Primary maternal infection often results in virus transmission, and symptomatic babies can have permanent neurological deficiencies and deafness. Congenital infection can also lead to intrauterine growth restriction, a defect in placental transport. HCMV replicates in primary cytotrophoblasts (CTBs, the specialized cells of the placenta, and inhibits differentiation/invasion. Human trophoblast progenitor cells (TBPCs give rise to the mature cell types of the chorionic villi, CTBs and multi-nucleated syncytiotrophoblasts (STBs. Here we report that TBPCs are fully permissive for pathogenic and attenuated HCMV strains. Studies with a mutant virus lacking a functional pentamer complex (gH/gL/pUL128-131A showed that virion entry into TBPCs is independent of the pentamer. In addition, infection is blocked by a potent human neutralizing monoclonal antibody (mAb, TRL345, reactive with glycoprotein B (gB, but not mAbs to the pentamer proteins pUL130/pUL131A. Functional studies revealed that neutralization of infection preserved the capacity of TBPCs to differentiate and assemble into trophospheres composed of CTBs and STBs in vitro. Our results indicate that mAbs to gB protect trophoblast progenitors of the placenta and could be included in antibody treatments developed to suppress congenital infection and prevent disease.

  7. β2-Glycoprotein I/IgA Immune Complexes: A Marker to Predict Thrombosis After Renal Transplantation in Patients With Antiphospholipid Antibodies.

    Science.gov (United States)

    Serrano, Manuel; Martínez-Flores, José A; Pérez, Dolores; García, Florencio; Cabrera, Oscar; Pleguezuelo, Daniel; Paz-Artal, Estela; Morales, José M; González, Esther; Serrano, Antonio

    2017-05-16

    Antiphospholipid syndrome is characterized by recurrent thrombosis and gestational morbidity in patients with antiphospholipid autoantibodies (aPLs). Predictive value of the presence of aPLs is low, and new markers are necessary to identify aPL carriers at higher risk and take preventive measures on them. The presence of circulating immune complexes of IgA bound to β 2 -glycoprotein I (B2A-CIC) has been associated with occurrence of acute thrombotic events. In this work we study its possible predictive value for the appearance of acute thrombotic events in patients who are going to undergo transplant surgery, a well-known trigger of acute thrombotic events in aPL carriers. We performed a follow-up study based on the Magnum 12+12 Cohort of patients who received a kidney transplant (n=1339). Three groups were established: group 1 patients who were positive for IgA anti-β 2 -glycoprotein I (aB2GP1) and B2A-CIC (n=125); group 2 patients who were positive only for IgA aB2GP1 (n=240); and control group, patients who were negative for IgA aB2GP1 (n=974). Levels of autoantibodies and B2A-CIC were quantified immediately before the transplant surgery and patients were followed up for 6 months. In group 1, 46.4% of patients experienced any type of thrombosis versus 10.4% in group 2 ( P thrombosis in group 1 (31.2%) was significantly higher than that observed in group 2 (3.3%, P thrombosis (hazard ratio, 6.72; 95% confidence interval, 4.81-9.37) and, prominently, for graft thrombosis (hazard ratio, 14.75; 95% confidence interval, 9.11-23.89). No significant differences were found between B2A-CIC-negative and control group patients. The presence of B2A-CIC is a predictor of acute thrombotic events. Patients who were positive for IgA aB2GP1 only are at risk of experiencing thrombosis if they are B2A-CIC positive. If they are B2A-CIC-negative patients, they have the same risk as the control group. Treatments to prevent acute thrombotic events should focus on B2A

  8. Use of synthetic peptides to represent surface-exposed epitopes defined by neutralizing dengue complex- and flavivirus group-reactive monoclonal antibodies on the native dengue type-2 virus envelope glycoprotein.

    Science.gov (United States)

    Falconar, Andrew K I

    2008-07-01

    The reactions of neutralizing monoclonal antibodies (mAbs) that defined dengue virus (DENV) complex, flavivirus subgroup or group neutralizing epitopes were tested against synthetic peptide sequences from domains I, II and III of the envelope (E) glycoproteins of different DENV-2 genotypes/strains. The DENV complex-reactive mAb identified the surface-exposed 304-GKFKV/IVKEIA-313 peptides and the DENV complex-conserved 393-KKGSSIGQ/KM-401 peptides in domain III, which were located adjacently in the native glycoprotein. Both flavivirus group-reactive mAbs reacted most strongly with fusion sequence peptides from domain II when they contained a cysteine (C) by glycine (G) substitution (underlined) (101-WGNGGGLFG-109) to represent the native rotated C side chain. The 393-401 sequence represents a newly identified epitope, present as a highly flexible coil located between the 385 and 393 cell-binding sequence and the 401 and 413 sequence involved in the E glycoprotein homo-trimer formation. The 101-109 sequence containing 105-C by G substitution and the 393-401 sequence are good candidates for diagnostic assays and cross-protection experiments.

  9. Characterization of a Novel 99mTc-Carbonyl Complex as a Functional Probe of MDR1 P-Glycoprotein Transport Activity

    Directory of Open Access Journals (Sweden)

    Mary Dyszlewski

    2002-01-01

    Full Text Available Multidrug resistance (MDR mediated by overexpression of MDR1 P-glycoprotein (Pgp is one of the best characterized barriers to chemotherapy in cancer patients. Furthermore, the protective function of Pgp-mediated efflux of xenobiotics in various organs has a profound effect on the bioavailability of drugs in general. Thus, there is an expanding requirement to noninvasively interrogate Pgp transport activity in vivo. We herein report the Pgp recognition properties of a novel 99mTc(I-tricarbonyl complex, [99mTc(CO3(MIBI3] + (Tc-CO-MIBI. Tc-CO-MIBI showed 60-fold higher accumulation in drug-sensitive KB 3–1 cells compared to colchicine-selected drug-resistant KB 8-5 cells. In KB 8-5 cells, tracer enhancement was observed with the potent MDR modulator LY335979 (EC50 = 62 nM. Similar behavior was observed using drug-sensitive MCF-7 breast adenocarcinoma cells and MCF-7/MDR1 stable transfectants, confirming that Tc-CO-MIBI is specifically excluded by overexpression of MDR1 Pgp. By comparison, net accumulation in control H69 lung tumor cells was 9-fold higher than in MDR-associated protein (MRP1-expressing H69AR cells, indicating only modest transport by MRP1. Biodistribution analysis following tail vein injection of Tc-CO-MIBI showed delayed liver clearance as well as enhanced brain uptake and retention in mdr1a/1b(−/− gene deleted mice versus wild-type mice, directly demonstrating that Tc-CO-MIBI is a functional probe of Pgp transport activity in vivo.

  10. Localization of MHC class II/human cartilage glycoprotein-39 complexes in synovia of rheumatoid arthritis patients using complex-specific monoclonal antibodies

    NARCIS (Netherlands)

    Steenbakkers, Peter G. A.; Baeten, Dominique; Rovers, Eric; Veys, Eric M.; Rijnders, Antonius W. M.; Meijerink, Jan; de Keyser, Filip; Boots, Annemieke M. H.

    2003-01-01

    Recently human cartilage gp-39 (HC gp-39) was identified as a candidate autoantigen in rheumatoid arthritis (RA). To further investigate the relevance of this Ag in RA, we have generated a set of five mAbs to a combination epitope of complexes of HC gp-39(263-275) and the RA-associated DR alpha beta

  11. HIV-1 Envelope Glycoprotein Biosynthesis, Trafficking, and Incorporation

    Science.gov (United States)

    Checkley, Mary Ann; Luttge, Benjamin G.; Freed, Eric O.

    2011-01-01

    The HIV-1 envelope (Env) glycoproteins play an essential role in the virus replication cycle by mediating the fusion between viral and cellular membranes during the entry process. The Env glycoproteins are synthesized as a polyprotein precursor, gp160, that is cleaved by cellular proteases to the mature surface glycoprotein gp120 and the transmembrane glycoprotein gp41. During virus assembly the gp120/gp41 complex is incorporated as heterotrimeric spikes into the lipid bilayer of nascent virions. These gp120/gp41 complexes then initiate the infection process by binding receptor and co-receptor on the surface of target cells. Much is currently known about the HIV-1 Env glycoprotein trafficking pathway and the structure of gp120 and the extracellular domain of gp41. However, the mechanism by which the Env glycoprotein complex is incorporated into virus particles remains incompletely understood. Genetic data support a major role for the cytoplasmic tail of gp41 and the matrix domain of Gag in Env glycoprotein incorporation. Still to be defined are the identities of host cell factors that may promote Env incorporation, and the role of specific membrane microdomains in this process. Here we review our current understanding of HIV-1 Env glycoprotein trafficking and incorporation into virions. PMID:21762802

  12. Early percutaneous coronary intervention, platelet inhibition with eptifibatide, and clinical outcomes in patients with acute coronary syndromes. PURSUIT Investigators

    NARCIS (Netherlands)

    N.S. Kleiman (Neal); D.V. Cokkinos (Dennis); M.L. Simoons (Maarten); R.A. Harrington (Robert Alex); R.M. Califf (Robert); E.J. Topol (Eric); A.M. Lincoff (Michael); G.C. Flaker; K.S. Pieper (Karen); R.G. Wilcox (Robert); L.G. Berdan (Lisa); T.J. Lorenz; H. Boersma (Eric)

    2000-01-01

    textabstractBACKGROUND: Platelet glycoprotein (GP) IIb/IIIa antagonists prevent the composite end point of death or myocardial infarction (MI) in patients with acute coronary syndromes. There is uncertainty about whether this effect is confined to patients who have percutaneous

  13. Effects of cobalt-chromium everolimus eluting stents or bare metal stent on fatal and non-fatal cardiovascular events

    DEFF Research Database (Denmark)

    Valgimigli, Marco; Sabaté, Manel; Kaiser, Christoph

    2014-01-01

    coronary syndrome v stable coronary artery disease), diabetes mellitus, female sex, use of glycoprotein IIb/IIIa inhibitors, and up to one year v longer duration treatment with dual antiplatelets. CONCLUSIONS: This meta-analysis offers evidence that compared with bare metal stents the use of cobalt...

  14. Pre-hospital administration of tirofiban in diabetic patients with ST-elevation myocardial infarction undergoing primary angioplasty: a sub-analysis of the On-Time 2 trial.

    NARCIS (Netherlands)

    Timmer, J.R.; Berg, J.; Heestermans, A.A.; Dill, T.; Werkum, J.W. van; Dambrink, J.H.; Suryapranata, H.; Ottervanger, J.P.; Hamm, C.; Hof, A.W. van 't

    2010-01-01

    AIMS: Glycoprotein IIb/IIIa blocking agents seem to improve percutaneous coronary intervention (PCI) results in patients with ST-elevation myocardial infarction (STEMI). We aimed to compare the effect of pre-hospital administration of tirofiban in STEMI patients with and without diabetes mellitus

  15. Disagreements between central clinical events committee and site investigator assessments of myocardial infarction end-points in an international clinical trial: review of the PURSUIT study

    NARCIS (Netherlands)

    K.W. Mahaffey (Kenneth); R.A. Harrington (Robert Alex); N.S. Kleiman (Neal); L.G. Berdan (Lisa); B.S. Crenshaw (Brian); B.E. Tardiff (Barbara); C.B. Granger (Christopher); I. DeJong (Ingrid); M. Bhapkar (Manju); P. Widimsky (Petr); R. Corbalon (Ramón); K.L. Lee (Kerry); J.W. Deckers (Jaap); M.L. Simoons (Maarten); E.J. Topol (Eric); R.M. Califf (Robert); K.M. Akkerhuis (Martijn)

    2001-01-01

    textabstractAbstract: Background Limited information has been published regarding how specific processes for event adjudication can affect event rates in trials. We reviewed nonfatal myocardial infarctions (MIs) reported by site investigators in the international Platelet Glycoprotein IIb/IIIa in

  16. Disagreements between central clinical events committee and site investigator assessments of myocardial infarction end- points in an international clinical trial: Review of the PURSUIT study

    NARCIS (Netherlands)

    K.W. Mahaffey (Kenneth); R.A. Harrington (Robert Alex); K.M. Akkerhuis (Martijn); N.S. Kleiman (Neal); L.G. Berdan (Lisa); B.S. Crenshaw (Brian); B.E. Tardiff (Barbara); C.B. Granger (Christopher); I. DeJong (Ingrid); M. Bhapkar (Manju); P. Widimsky (Petr); R. Corbalon (Ramón); K.L. Lee (Kerry); J.W. Deckers (Jaap); M.L. Simoons (Maarten); E.J. Topol (Eric); R.M. Califf (Robert)

    2001-01-01

    textabstractBackground. Limited information has been published regarding how specific processes for event adjudication can affect event rates in trials. We reviewed nonfatal myocardial infarctions (MIs) reported by site investigators in the international Platelet Glycoprotein IIb/IIIa in Unstable

  17. Early clinical outcomes as a function of use of newer oral P2Yinhibitors versus clopidogrel in the EUROMAX trial

    DEFF Research Database (Denmark)

    Huber, Kurt; Ducrocq, Gregory; Hamm, Christian W

    2017-01-01

    prehospital bivalirudin with heparin with optional glycoprotein IIb/IIIa inhibitor treatment in patients with ST-segment elevation myocardial infarction triaged to pPCI. Choice of P2Y12inhibitor was at the investigator's discretion. In a prespecified analysis, we compared event rates with clopidogrel...

  18. Risk of stroke associated with abciximab among patients undergoing percutaneous coronary intervention

    NARCIS (Netherlands)

    K.M. Akkerhuis (Martijn); A.M. Lincoff (Michael); J.E. Tcheng (James); K. Anderson (Keaven); C. Balog; R.M. Califf (Robert); E.J. Topol (Eric); M.L. Simoons (Maarten); J.W. Deckers (Jaap); H. Boersma (Eric)

    2001-01-01

    textabstractCONTEXT: Abciximab, a potent inhibitor of the platelet glycoprotein IIb/IIIa receptor, reduces thrombotic complications in patients undergoing percutaneous coronary intervention (PCI). Because of its potent inhibition of platelet aggregation, the effect of abciximab on risk of stroke is

  19. HIV-1 envelope glycoprotein

    Science.gov (United States)

    Caulfield, Michael; Cupo, Albert; Dean, Hansi; Hoffenberg, Simon; King, C. Richter; Klasse, P. J.; Marozsan, Andre; Moore, John P.; Sanders, Rogier W.; Ward, Andrew; Wilson, Ian; Julien, Jean-Philippe

    2017-08-22

    The present application relates to novel HIV-1 envelope glycoproteins, which may be utilized as HIV-1 vaccine immunogens, and antigens for crystallization, electron microscopy and other biophysical, biochemical and immunological studies for the identification of broad neutralizing antibodies. The present invention encompasses the preparation and purification of immunogenic compositions, which are formulated into the vaccines of the present invention.

  20. Glycoprotein and proteoglycan techniques

    International Nuclear Information System (INIS)

    Beeley, J.G.

    1985-01-01

    The aim of this book is to describe techniques which can be used to answer some of the basic questions about glycosylated proteins. Methods are discussed for isolation, compositional analysis, and for determination of the primary structure of carbohydrate units and the nature of protein-carbohydrate linkages of glycoproteins and proteoglycans. High resolution NMR is considered, as well as radioactive labelling techniques. (Auth.)

  1. On the use of abciximab in percutaneous coronary intervention

    DEFF Research Database (Denmark)

    Iversen, Allan

    2011-01-01

    on angiogram (complex versus simple) and use of abciximab. Follow-up was 1 year. The endpoints were: mortality, TVR, MI, and the composite of the three. Results: • Patients with diabetes who experienced ACS and who were treated with PCI and abciximab had their mortality and need of TVR reduced significantly......Introduction: The present thesis ´On the use of abciximab in percutaneous coronary intervention´ is based on 6 papers concerning the glycoprotein IIb/IIIa inhibitor, abciximab. The thesis is divided into 2 sections. One section concerning a randomized trial comparing intracoronary (IC......) with intravenous (IV) abciximab in patients with ST-segment elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (pPCI), and one section concerning results from 3 registry studies on the effect of abciximab in distinct subgroups of patients with acute coronary syndrome...

  2. On the use of abciximab in percutaneous coronary intervention

    DEFF Research Database (Denmark)

    Iversen, Allan

    2011-01-01

    as a review (paper I). Objectives and methods: In order to investigate if IC administration of bolus abciximab indeed was superior to IV administration, we set up a randomized, open-label, single-center trial. We randomized 355 STEMI patients treated with pPCI between 2006 and 2008 to receive either IC or IV...... (ACS). Optimal administration route of abciximab. A randomized study Background: The glycoprotein IIb/IIIa inhibitor, abciximab, is used as an adjuvant anti-platelet therapy in PCI-treated patients suffering from ACS. A subgroup of patients with ACS is those with STEMI treated with p...... might benefit more than others, and that abciximab might even be harmful in yet others. Although data are not consistent, suggestions have been made, that risk factors, such as diabetes, age and complexity of the coronary lesions might influence the efficacy of abciximab. Objectives and methods...

  3. Detection of circulating immune complexes of human IgA and beta 2 glycoprotein I in patients with antiphospholipid syndrome symptomatology.

    Science.gov (United States)

    Martínez-Flores, José A; Serrano, Manuel; Pérez, Dolores; Lora, David; Paz-Artal, Estela; Morales, José M; Serrano, Antonio

    2015-07-01

    Patients with antiphospholipid syndrome (APS) have a hypercoagulable condition associated with the presence of antiphospholipid autoantibodies (aPL). Consensus antibodies for diagnosis are lupus anticoagulant, anti-beta2 glycoprotein I (B2GPI) and anticardiolipin (IgG or IgM). Circulating immunocomplexes (CIC) of B2GPI associated with IgM or IgG were reported. Isolated IgA aB2GPI antibodies have achieved high diagnostic value although specific CIC of B2GPI bounded to IgA (B2A-CIC) has still not been described. CIC detection assays are mainly based on interaction with complement and are not appropriate to detect B2A-CIC because IgA does not fix complement using the classical pathway. Sera from healthy blood donors (N= 247) and from patients with thrombosis background and isolate positive for IgA aB2GPI (N = 68) were studied in a case-control study. Two methods were applied, these being a capture ELISA to quantify specific B2A-CIC and quantification of total IgA anti-B2GPI after dissociating CIC. B2A-CIC values in APS-patients were 19.27 ± 2.6 AU vs 6.1 ± 0.4 AU in blood donors (p < 0.001). There were 36.4% B2A-CIC positive patients (cutoff 21 AU) versus 5.5% in blood donors (p < 0.001). Dissociated IgA aB2GPI levels (total IgA aB2GPI) were 146.8 ± 10.8 IU/mL in patients vs. 22.4 IU/mL in controls (p < 0.001). B2A-CIC was independent of B2GPI and autoantibodies IgA aB2GPI serum levels. B2A-CIC can be identified and quantified in an easy and reproducible manner using two complement-independent methods. The use of these tests in prospective studies will allow better understanding of the prognosis and outcome of patients. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Structural mimicry of the dengue virus envelope glycoprotein revealed by the crystallographic study of an idiotype-anti-idiotype Fab complex.

    Science.gov (United States)

    Wong, Yee Hwa; Goh, Boon Chong; Lim, She Yah; Teo, En Wei; Lim, Angeline P C; Dedon, Pete C; Hanson, Brendon J; MacAry, Paul A; Lescar, Julien

    2017-06-21

    A detailed understanding of the fine specificity of serotype-specific human antibodies is vital for the development and evaluation of new vaccines for pathogenic Flaviviruses such as Dengue virus (DENV) and Zika virus. In this study, we thoroughly characterize the structural footprint of an anti-idiotype antibody (E1) specific for a potent, fully human DENV serotype 1-specific antibody termed HM14c10, derived from a recovered patient. The crystal structure at a resolution of 2.5 Å of a complex between the Fab fragments of E1 and HM14c10 provides the first detailed molecular comparison of an anti-idiotype paratope specific for a human antibody with its analogous epitope- a discontinuous quaternary structure located at the surface of the viral particle that spans adjacent envelope (E) proteins. This comparison reveals that the footprints left by E1 and E on HM14c10 largely overlap, explaining why formation of the binary complexes are mutually exclusive. Structural mimicry of the DENV E epitope by the E1 combining site is achieved via the formation of numerous interactions with heavy chain CDRs of HM14c10, while fewer interactions are observed with its light chain, compared to the E protein. We show that E1 can be utilized to detect HM14c10-like antibodies in sera from patients recovered from a DENV-1 infection suggesting that this is a public (common) idiotype. These data demonstrate the utility of employing an anti-idiotype antibody to monitor a patient's specific immune responses and suggest routes for improvement of E 'mimicry' by E1 through increasing its recognition of the FabHM14c10 light chain CDRs. IMPORTANCE A chimeric yellow fever/dengue live-attenuated tetravalent vaccine is now marketed. Dengue remains a significant public health problem, because protection conferred by this vaccine is uneven against the four circulating serotypes. Reliable tools must be developed to measure the immune response of individuals exposed to DENV, either via viral infection

  5. Human periodontal ligament stem cells suppress T-cell proliferation via down-regulation of non-classical major histocompatibility complex-like glycoprotein CD1b on dendritic cells.

    Science.gov (United States)

    Shin, C; Kim, M; Han, J-A; Choi, B; Hwang, D; Do, Y; Yun, J-H

    2017-02-01

    Periodontal ligament stem cells (PDLSCs) from the periodontal ligament tissue were recently identified as mesenchymal stem cells (MSCs). The capabilities of PDLSCs in periodontal tissue or bone regeneration have been reported, but their immunomodulatory role in T-cell immune responses via dendritic cells (DCs), known as the most potent antigen-presenting cell, has not been studied. The aim of this study is to understand the immunological function of homogeneous human STRO-1 + CD146 + PDLSCs in DC-mediated T-cell immune responses to modulate the periodontal disease process. We utilized highly purified (> 95%) human STRO-1 + CD146 + PDLSCs and human bone marrow mesenchymal stem cells (BMSCs). Each stem cell was co-cultured with human monocyte-derived DCs in the presence of lipopolysaccharide isolated from Porphyromonas gingivalis, a major pathogenic bacterium responsible for periodontal disease, in vitro to examine the immunological effect of each stem cell on DCs and DC-mediated T-cell proliferation. We discovered that STRO-1 + CD146 + PDLSCs, as well as BMSCs, significantly decreased the level of non-classical major histocompatibility complex glycoprotein CD1b on DCs, resulting in defective T-cell proliferation, whereas most human leukocyte antigens and the co-stimulatory molecules CD80 and CD86 in/on DCs were not significantly affected by the presence of BMSCs or STRO-1 + CD146 + PDLSCs. This study unveiled an immunomodulatory role of STRO-1 + CD146 + PDLSCs in negatively regulating DC-mediated T-cell immune responses, demonstrating their potential to be utilized in promising new stem cell therapies. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Processing of virus-specific glycoproteins of varicella zoster virus

    Energy Technology Data Exchange (ETDEWEB)

    Namazue, J.; Campo-Vera, H.; Kitamura, K.; Okuno, T.; Yamanishi, K.

    1985-05-01

    Monoclonal antibodies to varicella zoster virus (VZV) glycoproteins were used to study the processing of three glycoproteins with molecular weights of 83K-94K (gp 2), 64K (gp 3), and 55K (gp 5). Immunoprecipitation experiments performed with VZV-infected cells, pulse labeled with (/sup 3/H)glucosamine in the presence of tunicamycin, suggest that O-linked oligosaccharide is present on the glycoprotein of gp 2. Use of the enzyme endo-beta-N-acetylglucosaminidase H revealed that the fully processed form of gp 3 had high-mannose type and that of gp 5 had only complex type of N-linked oligosaccharides. Experiments with monensin suggest that the precursor form (116K) of gp 3 is cleaved during the processing from Golgi apparatus to cell surface membrane. The extension of O-linked oligosaccharide chain and the complex type of N-linked oligosaccharide chains also occurs during this processing.

  7. Processing of virus-specific glycoproteins of varicella zoster virus

    International Nuclear Information System (INIS)

    Namazue, J.; Campo-Vera, H.; Kitamura, K.; Okuno, T.; Yamanishi, K.

    1985-01-01

    Monoclonal antibodies to varicella zoster virus (VZV) glycoproteins were used to study the processing of three glycoproteins with molecular weights of 83K-94K (gp 2), 64K (gp 3), and 55K (gp 5). Immunoprecipitation experiments performed with VZV-infected cells, pulse labeled with [ 3 H]glucosamine in the presence of tunicamycin, suggest that O-linked oligosaccharide is present on the glycoprotein of gp 2. Use of the enzyme endo-beta-N-acetylglucosaminidase H revealed that the fully processed form of gp 3 had high-mannose type and that of gp 5 had only complex type of N-linked oligosaccharides. Experiments with monensin suggest that the precursor form (116K) of gp 3 is cleaved during the processing from Golgi apparatus to cell surface membrane. The extension of O-linked oligosaccharide chain and the complex type of N-linked oligosaccharide chains also occurs during this processing

  8. Cancer Biomarker Discovery: Lectin-Based Strategies Targeting Glycoproteins

    Directory of Open Access Journals (Sweden)

    David Clark

    2012-01-01

    Full Text Available Biomarker discovery can identify molecular markers in various cancers that can be used for detection, screening, diagnosis, and monitoring of disease progression. Lectin-affinity is a technique that can be used for the enrichment of glycoproteins from a complex sample, facilitating the discovery of novel cancer biomarkers associated with a disease state.

  9. Direct chemical modification and voltammetric detection of glycans in glycoproteins

    Czech Academy of Sciences Publication Activity Database

    Trefulka, Mojmír; Paleček, Emil

    2014-01-01

    Roč. 48, NOV2014 (2014), s. 52-55 ISSN 1388-2481 R&D Projects: GA ČR(CZ) GAP301/11/2055 Institutional support: RVO:68081707 Keywords : Glycoproteins * Chemical modification * Os(VI)L complexes Subject RIV: BO - Biophysics Impact factor: 4.847, year: 2014

  10. The glycoprotein of measles virus

    International Nuclear Information System (INIS)

    Anttonen, O.; Jokinen, M.; Salmi, A.; Vainionpaeae, R.; Gahmberg, C.G.

    1980-01-01

    Measles virus was propagated in VERO cells and purified from the culture supernatants by two successive tartrate-density-gradient centrifugations. Surface carbohydrates were labelled both in vitro and in vivo with 3 H after treatment with galactose oxidase/NaB 3 H 4 or with [ 3 H]glucosamine. The major labelled glycoprotein in measles virions had a mol.wt. of 79000. After labelling with periodate/NaB 3 H 4 , which would result in specific labelling of sialic acid residues, the 79000-mol.wt. glycoprotein was very weakly labelled. This suggested that there is no or a very low amount of sialic acid in the virions. Further analysis of the glycoprotein showed that galactose is the terminal carbohydrate unit in the oligosaccharide, and the molecular weight of the glycopeptide obtained after Pronase digestion is about 3000. The oligosaccharide is attached to the polypeptide through an alkali-stable bond, indicating a N-glycosidic asparagine linkage. (author)

  11. Recent Progress in Electrochemical Biosensors for Glycoproteins

    Directory of Open Access Journals (Sweden)

    Uichi Akiba

    2016-12-01

    Full Text Available This review provides an overview of recent progress in the development of electrochemical biosensors for glycoproteins. Electrochemical glycoprotein sensors are constructed by combining metal and carbon electrodes with glycoprotein-selective binding elements including antibodies, lectin, phenylboronic acid and molecularly imprinted polymers. A recent trend in the preparation of glycoprotein sensors is the successful use of nanomaterials such as graphene, carbon nanotube, and metal nanoparticles. These nanomaterials are extremely useful for improving the sensitivity of glycoprotein sensors. This review focuses mainly on the protocols for the preparation of glycoprotein sensors and the materials used. Recent improvements in glycoprotein sensors are discussed by grouping the sensors into several categories based on the materials used as recognition elements.

  12. Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

    DEFF Research Database (Denmark)

    Vester-Christensen, Malene B; Halim, Adnan; Joshi, Hiren Jitendra

    2013-01-01

    The metazoan O-mannose (O-Man) glycoproteome is largely unknown. It has been shown that up to 30% of brain O-glycans are of the O-Man type, but essentially only alpha-dystroglycan (α-DG) of the dystrophin-glycoprotein complex is well characterized as an O-Man glycoprotein. Defects in O-Man glycos......The metazoan O-mannose (O-Man) glycoproteome is largely unknown. It has been shown that up to 30% of brain O-glycans are of the O-Man type, but essentially only alpha-dystroglycan (α-DG) of the dystrophin-glycoprotein complex is well characterized as an O-Man glycoprotein. Defects in O...

  13. Proteolytic Processing of the Human Immunodeficiency Virus Envelope Glycoprotein Precursor Decreases Conformational Flexibility

    OpenAIRE

    Haim, Hillel; Salas, Ignacio; Sodroski, Joseph

    2013-01-01

    The mature envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) virions is derived by proteolytic cleavage of a trimeric gp160 glycoprotein precursor. Remarkably, proteolytic processing of the HIV-1 Env precursor results in changes in Env antigenicity that resemble those associated with glutaraldehyde fixation. Apparently, proteolytic processing of the HIV-1 Env precursor decreases conformational flexibility of the Env trimeric complex, differentiall...

  14. Glycoprotein Ib and glycoprotein IX in human platelets are acylated with palmitic acid through thioester linkages

    International Nuclear Information System (INIS)

    Muszbek, L.; Laposata, M.

    1989-01-01

    The glycoprotein (GP) Ib-IX complex is a major component of the platelet membrane which mediates adhesion of platelets to exposed subendothelium. GP Ib is a heterodimer with a large alpha chain (Mr = 135,000-145,000) and small beta chain (Mr = 22,000-27,000) linked by a disulfide bond(s). GP Ib is bound in a noncovalent 1:1 complex with GP IX (Mr = 17,000-22,000). We labeled isolated human platelets with [3H] palmitate or surface-labeled platelet membrane glycoproteins with sodium periodate-[3H]sodium borohydride and immunoprecipitated the GP Ib-IX complex from radiolabeled platelet lysates using a mouse monoclonal antibody (SZ.1) which recognizes the intact complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitates from [3H]palmitate-labeled platelets revealed two radiolabeled bands under reducing conditions at 24 and 19 kDa and two bands under nonreducing conditions at 170 and 19 kDa. As demonstrated by the parallel analysis of immunoprecipitates from periodate-[3H]sodium borohydride-labeled platelets, the [3H]palmitate-labeled bands obtained under reducing conditions corresponded to GP Ib beta and GP IX and the ones obtained under nonreducing conditions to intact GP Ib and GP IX, respectively. Using alkaline methanolysis followed by high pressure liquid chromatography analysis of the methanolysis products, we demonstrated that the radioactivity associated with the GP Ib-IX complex from [3H]palmitate-labeled platelets was, in fact, covalently bound [3H]palmitate in ester linkage to protein. The protein-fatty acid linkage was also disrupted by hydroxylamine at neutral pH. Thus, this study demonstrates that GP Ib beta and GP IX in human platelets are both fatty acid-acylated with palmitate through thioester linkages

  15. Fbs1 protects the malfolded glycoproteins from the attack of peptide:N-glycanase

    International Nuclear Information System (INIS)

    Yamaguchi, Yoshiki; Hirao, Takeshi; Sakata, Eri; Kamiya, Yukiko; Kurimoto, Eiji; Yoshida, Yukiko; Suzuki, Tadashi; Tanaka, Keiji; Kato, Koichi

    2007-01-01

    Fbs1 is a cytosolic lectin putatively operating as a chaperone as well as a substrate-recognition subunit of the SCF Fbs1 ubiquitin ligase complex. To provide structural and functional basis of preferential binding of Fbs1 to unfolded glycoproteins, we herein characterize the interaction of Fbs1 with a heptapeptide carrying Man 3 GlcNAc 2 by nuclear magnetic resonance (NMR) spectroscopy and other biochemical methods. Inspection of the NMR data obtained by use of the isotopically labeled glycopeptide indicated that Fbs1 interacts with sugar-peptide junctions, which are shielded in native glycoprotein, in many cases, but become accessible to Fbs1 in unfolded glycoproteins. Furthermore, Fbs1 was shown to inhibit deglycosylation of denatured ribonuclease B by a cytosolic peptide:N-glycanase (PNGase). On the basis of these data, we suggest that Fbs1 captures malfolded glycoproteins, protecting them from the attack of PNGase, during the chaperoning or ubiquitinating operation in the cytosol

  16. Isolation of glycoproteins from brown algae.

    OpenAIRE

    Surendraraj, Alagarsamy; Farvin Koduvayur Habeebullah , Sabeena; Jacobsen, Charlotte

    2015-01-01

    The present invention relates to a novel process for the isolation of unique anti-oxidative glycoproteins from the pH precipitated fractions of enzymatic extracts of brown algae. Two brown seaweeds viz, Fucus serratus and Fucus vesiculosus were hydrolysed by using 3 enzymes viz, Alcalase, Viscozyme and Termamyl and the glycoproteins were isolated from these enzyme extracts.

  17. Outcomes of patients with coronary artery perforation complicating percutaneous coronary intervention and correlations with the type of adjunctive antithrombotic therapy: pooled analysis from REPLACE-2, ACUITY, and HORIZONS-AMI trials.

    Science.gov (United States)

    Doll, Jacob A; Nikolsky, Eugenia; Stone, Gregg W; Mehran, Roxana; Lincoff, A Michael; Caixeta, Adriano; McLaurin, Brent; Cristea, Ecaterina; Fahy, Martin; Kesanakurthy, Vijaya; Lansky, Alexandra J

    2009-10-01

    The lack of a specific counteragent to bivalirudin may complicate the management of patients with coronary artery (CA) perforation during percutaneous coronary intervention (PCI). Assess outcomes of patients with CA perforation from three PCI trials comparing intravenous bivalirudin with provisional glycoprotein (GP) IIb/IIIa inhibition versus unfractionated heparin (UFH) plus GP IIb/IIIa. A pooled analysis of patients treated with PCI in three randomized trials including REPLACE-2, ACUITY, and HORIZONS-AMI. Among a total of 12,921 patients, CA perforation occurred in 35 patients (0.27%). By multivariable analysis, baseline creatinine clearance was the only independent predictor of CA perforation (per 10 mL/min decrease, odds ratio [95% confidence interval]= 1.28 [1.11, 1.47], P = 0.0007). At 30 days, patients with versus without CA perforation had significantly (all P values therapy of bivalirudin monotherapy was not associated with worse outcomes compared to treatment with UFH plus GP IIb/IIIa inhibitors.

  18. The effects of platelet inhibitors on blood use in cardiac surgery.

    Science.gov (United States)

    Lee, Leonard Y; DeBois, William; Krieger, Karl H; Girardi, Leonard N; Russo, Laura; McVey, James; Ko, Wilson; Altorki, Nasser K; Brodman, Richard A; Isom, O Wayne

    2002-01-01

    Platelet inhibition via glycoprotein (GP) IIb/IIIa receptor antagonists has greatly reduced the need for emergent cardiac surgery. However, this change has come at a cost to both the patient and the cardiac surgical team in terms of increased bleeding risk. Current guidelines for patients requiring coronary artery bypass surgery include: 1) cessation of GP IIb/IIIa inhibitor; 2) delay of surgery for up to 12 h if abciximab, tirofiban, or eptafibitide is used; 3) utilization of ultrafiltration via zero balance technique; 4) maintenance of standard heparin dosing despite elevated bleeding times; and 5) transfusion of platelets as needed, rather than prophylactically. These agents present cardiac surgery teams with increased risk during CABG, although overall risk may be diminished by the substantial benefits to patients with acute coronary syndromes and percutaneous interventions, i.e., reduced infarction rates and improved vessel patency. With judicious planning, urgent coronary artery bypass can be safely performed on patients who have been treated with GP IIb/IIIa receptor inhibitors.

  19. Platelet Glycoprotein Ib-IX and Malignancy

    Science.gov (United States)

    2010-09-01

    whether adjunct anti-GP Ib-IX therapy could benefit the breast cancer patient with malignant disease. Body Below we list the 3 Specific Aims from our...Platelet Glycoprotein Ib-IX and Malignancy PRINCIPAL INVESTIGATOR: Jerry Ware, Ph.D...AND SUBTITLE Platelet Glycoprotein Ib-IX and Malignancy 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-08-1-0576 5c

  20. Caracterização da ativação plaquetária nos concentrados de plaquetas por citometria de fluxo

    Directory of Open Access Journals (Sweden)

    Landi Evaldo P.

    2003-01-01

    Full Text Available Flow cytometric characterization of platelet membrane glycoproteins expression and procoagulant surface is a sensitive technique to identify platelet activation in platelet concentrates. Platelet activation could contribute to platelet storage lesion reducing platelet viability and its hemostatic function. Detection of activation in platelet concentrates may help to determine the mechanism by which platelets activate during preparation and storage. It may also suggest interventions to prevent and reduce the platelet storage lesion. A monoclonal antibody panel that evaluate glycoproteins IIb-IIIa and Ib-IX migration, platelet degranulation and platelet procoagulant surface would provide more information about platelet activation than activation-dependent monoclonal antibodies alone.

  1. Functional platelet defects in children with severe chronic ITP as tested with 2 novel assays applicable for low platelet counts.

    Science.gov (United States)

    van Bladel, Esther R; Laarhoven, Annemieke G; van der Heijden, Laila B; Heitink-Pollé, Katja M; Porcelijn, Leendert; van der Schoot, C Ellen; de Haas, Masja; Roest, Mark; Vidarsson, Gestur; de Groot, Philip G; Bruin, Marrie C A

    2014-03-06

    Immune thrombocytopenia (ITP) is an autoimmune disease with a complex heterogeneous pathogenesis and a bleeding phenotype that is not necessarily correlated to platelet count. In this study, the platelet function was assessed in a well-defined cohort of 33 pediatric chronic ITP patients. Because regular platelet function test cannot be performed in patients with low platelet counts, 2 new assays were developed to determine platelet function: first, the microaggregation test, measuring in platelets isolated from 10 mL of whole blood the platelet potential to form microaggregates in response to an agonist; second, the platelet reactivity assay, measuring platelet reactivity to adenosine diphosphate (ADP), convulxin (CVX), and thrombin receptor activator peptide in only 150 μL of unprocessed whole blood. Patients with a severe bleeding phenotype demonstrated a decreased aggregation potential upon phorbol myristate acetate stimulation, decreased platelet degranulation following ADP stimulation, and a higher concentration of ADP and CVX needed to activate the glycoprotein IIbIIIa complex compared with patients with a mild bleeding phenotype. In conclusion, here we have established 2 functional tests that allow for evaluation of platelet function in patients with extremely low platelet counts (<10(9)). These tests show that platelet function is related to bleeding phenotype in chronic ITP.

  2. Bivalirudin is superior to heparins alone with bailout GP IIb/IIIa inhibitors in patients with ST-segment elevation myocardial infarction transported emergently for primary percutaneous coronary intervention

    DEFF Research Database (Denmark)

    Zeymer, Uwe; van 't Hof, Arnoud; Adgey, Jennifer

    2014-01-01

    in comparison with heparins only with GPI used as bailout. METHODS AND RESULTS: In the EUROMAX study, 2198 patients with ST-segment elevation myocardial infarction (STEMI) were randomized during transport for primary PCI to bivalirudin or to heparins with optional GPI. Primary and principal outcome...... outcome death and major bleeding occurred in 5.1% with bivalirudin, 7.6% with heparin plus routine GPI (HR 0.67 and 95% CI 0.46-0.97, P = 0.034), and 9.8% with heparins plus bailout GPI (HR 0.52 and 95% CI 0.35-0.75, P = 0.006). Following adjustment by logistic regression, bivalirudin was still associated...

  3. Targeting HIV-1 Envelope Glycoprotein Trimers to B Cells by Using APRIL Improves Antibody Responses

    NARCIS (Netherlands)

    Melchers, Mark; Bontjer, Ilja; Tong, Tommy; Chung, Nancy P. Y.; Klasse, Per Johan; Eggink, Dirk; Montefiori, David C.; Gentile, Maurizio; Cerutti, Andrea; Olson, William C.; Berkhout, Ben; Binley, James M.; Moore, John P.; Sanders, Rogier W.

    2012-01-01

    An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these

  4. Modification-specific proteomic analysis of glycoproteins in human body fluids by mass spectrometry

    DEFF Research Database (Denmark)

    Bunkenborg, Jakob; Hägglund, Per; Jensen, Ole Nørregaard

    2007-01-01

    -glycosylated proteins in body fluids and other complex samples. An approach for identification of N-glycosylated proteins and mapping of their glycosylation sites is described. In this approach, glycoproteins are initially selectively purified by lectin chromatography. Following tryptic digestion, glycopeptides...

  5. Glycoprotein biosynthesis by human normal platelets

    International Nuclear Information System (INIS)

    Rodriguez, P.; Bello, O.; Apitz-Castro, R.

    1987-01-01

    Incorporation of radioactive Man, Gal, Fuc, Glc-N, and NANA into washed human normal platelets and endogenous glycoproteins has been found. Both parameters were time dependent. Analysis of hydrolyzed labeled glycoproteins by paper chromatography revealed that the radioactive monosaccharide incubated with the platelets had not been converted into other sugars. Acid hydrolysis demonstrates the presence of a glycosidic linkage. All the effort directed to the demonstration of the existence of a lipid-sugar intermediate in intact human platelets yielded negative results for Man and Glc-N used as precursors. The incorporation of these sugars into glycoproteins is insensitive to bacitracin, suggesting no involvement of lipid-linked saccharides in the synthesis of glycoproteins in human blood platelets. The absence of inhibition of the glycosylation process in the presence of cycloheximide suggests that the sugars are added to proteins present in the intact platelets. These results support the contention that glycoprotein biosynthesis in human blood platelets observed under our experimental conditions is effected through direct sugar nucleotide glycosylation

  6. Detergent-Assisted Glycoprotein Capture: A Versatile Tool for In-Depth N-Glycoproteome Analysis.

    Science.gov (United States)

    Chen, Rui; Zou, Hanfa; Figeys, Daniel

    2016-06-03

    Large-scale N-glycoproteome studies have been hindered by poor solubility of hydrophobic membrane proteins and the complexity of proteome samples. Herein, we developed a detergent-assisted glycoprotein capture method to reduce these issues by conducting hydrazide chemistry-based glycoprotein capture in the presence of strong detergents such as sodium dodecyl sulfate and Triton X-100. The strong detergents helped to solubilize hydrophobic membrane proteins and then increased the access of hydrazide groups to oxidized glycoproteins, thus increasing the coverage of the N-glycoproteome. Compared with the conventional glycopeptide capture method, the detergent-assisted glycoprotein capture approach nearly doubled the number of N-glycosylation sites identified from HEK 293T cells with improved specificity. Application of this approach in the larger scale N-glycoproteomics analysis of the HEK 293T cell membrane led to the identification of 2253 unique N-glycosites from 953 proteins. Furthermore, the application of this approach to human serum resulted in the identification of 850 N-glycosylation sites without any immunodepletion or fractionation. Overall, the detergent-assisted glycoprotein capture method simplified the capture process, and it increased the number of sites observed on both hydrophobic membrane proteins and hydrophilic secreted proteins.

  7. Influence of O(2)-carrying plasma hemoprotein "albumin-heme" on complement system and platelet activation in vitro and physiological responses to exchange transfusion.

    Science.gov (United States)

    Komatsu, Teruyuki; Huang, Yubin; Wakamoto, Shinobu; Abe, Hideki; Fujihara, Mitsuhiro; Azuma, Hiroshi; Ikeda, Hisami; Yamamoto, Hisashi; Horinouchi, Hirohisa; Kobayashi, Koichi; Tsuchida, Eishun

    2007-06-15

    Recombinant human serum albumin (HSA) including the synthetic iron(II)-porphyrin (FeP), albumin-heme (HSA-FeP), is a unique O(2)-carrying plasma hemoprotein as a red blood cell substitute. We have investigated the possible influence of HSA-FeP on the complement system and platelet activation in vitro. The amounts of the serum complement titer CH(50) and terminal complement complex SC5b-9 of human blood serum, incubated with HSA-FeP (10, 20, and 40 vol %), were almost the same as those of the corresponding samples with HSA. The effect of HSA-FeP on the platelet reactivity has been demonstrated by conformational changes in the membrane glycoprotein IIb/IIIa and surface expression of an alpha-granule membrane protein P-selectin. Platelet activation in response to the ADP-stimulation was not influenced by the presence of HSA-FeP. It can be concluded that the albumin-heme solution does not facilitate the immunological reaction and platelet activation. Moreover, a 20% exchange transfusion with HSA-FeP into anesthetized rats has been performed to evaluate the circulation and blood parameters for 6 h. Time course changes in all parameters showed features identical to the control group (without infusion) and HSA group. (c) 2007 Wiley Periodicals, Inc.

  8. Intracoronary versus Intravenous eptifibatide during percutaneous coronary intervention for acute ST-segment elevation myocardial infarction; a randomized controlled trial.

    Science.gov (United States)

    Sanati, Hamid Reza; Zahedmehr, Ali; Firouzi, Ata; Farrashi, Melody; Amin, Kamyar; Peighambari, Mohammad Mehdi; Shakerian, Farshad; Kiani, Reza

    2017-10-01

    Although aspirin and clopidogrel seem to be quite enough during low risk percutaneous coronary intervention (PCI), the combination may need some reinforcement in complex situations such as primary PCI. By modifying the route and also the duration of administration, glycoprotein IIb/IIIa inhibitors might be a viable option. The aim of this study is to compare the benefits and disadvantages of three different methods of administration of eptifibatide in primary PCI population. Primary PCI candidates were randomized in three groups on which three different methods of administration of eptifibitide were tested: intravenous bolus injection followed by 12-h infusion (IV-IV), intracoronary bolus injection followed by intravenous infusion (IC-IV) and, only intracoronary bolus injection (IC). 99 patients were included in the present study. There was no significant difference among the three groups regarding all cause in hospital and one month mortality (p value = 0.99), re-myocardial infarction (p value = 0.89), post-PCI TIMI flow grade 3 (p value = 0.97), ST segment resolution (p value = 0.77) and peak troponin levels (p value = 0.82). The comparison of vascular access and major bleeding complications were not possible due to low events rate. By modifying the route of administration of eptifibitide, the clinical effect might be preserved without increasing the short-term mortality and procedural failure.

  9. Dual inhibition of PI3K and mTOR by VS-5584 suppresses thrombus formation.

    Science.gov (United States)

    Später, Thomas; Müller, Isabelle; Eichler, Hermann; Menger, Michael D; Laschke, Matthias W; Ampofo, Emmanuel

    2017-05-15

    VS-5584 is a highly selective dual kinase inhibitor which suppresses phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) activity. Because these kinases are crucially involved in primary hemostasis, we herein investigated the effect of this compound on thrombus formation in vitro and in vivo. Pretreatment of washed platelets (WP) or platelet-rich plasma (PRP) with VS-5584 inhibited the agonist-induced activation of surface glycoprotein complex (GP)IIb/IIIa and the upregulation of P-selectin. This was associated with a significantly reduced formation of platelet-leukocyte aggregates (PLA). VS-5584 further attenuated platelet aggregation and adhesion after agonist stimulation. In contrast, endothelial expression of intercellular adhesion molecule (ICAM)-1 and vascular cellular adhesion molecule (VCAM)-1 and secretion of von Willebrand Factor (vWF) were not affected by the dual kinase inhibitor. In vivo, VS-5584 inhibited photochemically induced thrombus formation as shown by a significantly prolonged time to complete vessel occlusion when compared to vehicle-treated controls. This was associated with an elevated tail vein bleeding time, indicating a potential hemorrhagic risk in VS-5584-treated mice. Taken together, these novel findings demonstrate that VS-5584 is a potent inhibitor of primary hemostasis targeting multiple platelet functions.

  10. Loss of fibrinogen receptors from the platelet surface during simulated extracorporeal circulation

    Energy Technology Data Exchange (ETDEWEB)

    Musial, J.; Niewiarowski, S.; Hershock, D.; Morinelli, T.A.; Colman, R.W.; Edmunds, L.H. Jr.

    1985-04-01

    In vitro recirculation of fresh human heparinized blood in an extracorporeal circuit with a membrane oxygenator decreased fibrinogen- induced platelet aggregation and diminished the number of fibrinogen receptors and glycoprotein IIb/IIIa (GPIIb/GPIIIa) antigenic sites on the platelet surface. In seven experiments, the mean +/- SD Km value for fibrinogen (i.e., molar concentration of fibrinogen required to cause 50% of the maximal rate of aggregation) was 1.58 x 10(-7) mol/L +/- 0.68 x 10(-7) mol/L. After recirculation, this value increased to 3.8 x 10(-7) mol/L +/- 1.94 x 10(-7) mol/L. The maximal aggregation rate of chymotrypsin-treated platelets decreased by 40% after 2 hours of recirculation. The number of fibrinogen receptors on platelets, which were treated with chymotrypsin after a recirculation, decreased from 41,370 +/- 24,000 to 13,230 +/- 10,230/platelet under the same conditions. The number of antigenic sites for monoclonal antibody reacting with GPIIb/GPIIIa complex of adenosine diphosphate-stimulated platelets decreased from 34,200 +/- 5,940 to 19,500 +/- 9,680/platelet after recirculation. Prostaglandin E1 (0. 3 mumol/L) in the perfusion circuit preserved the ability of platelets to react with fibrinogen. In conclusion, the loss of fibrinogen receptors from the surface of platelet membranes results from the interaction of platelets with the surfaces of perfusion circuits.

  11. Loss of fibrinogen receptors from the platelet surface during simulated extracorporeal circulation

    International Nuclear Information System (INIS)

    Musial, J.; Niewiarowski, S.; Hershock, D.; Morinelli, T.A.; Colman, R.W.; Edmunds, L.H. Jr.

    1985-01-01

    In vitro recirculation of fresh human heparinized blood in an extracorporeal circuit with a membrane oxygenator decreased fibrinogen- induced platelet aggregation and diminished the number of fibrinogen receptors and glycoprotein IIb/IIIa (GPIIb/GPIIIa) antigenic sites on the platelet surface. In seven experiments, the mean +/- SD Km value for fibrinogen (i.e., molar concentration of fibrinogen required to cause 50% of the maximal rate of aggregation) was 1.58 x 10(-7) mol/L +/- 0.68 x 10(-7) mol/L. After recirculation, this value increased to 3.8 x 10(-7) mol/L +/- 1.94 x 10(-7) mol/L. The maximal aggregation rate of chymotrypsin-treated platelets decreased by 40% after 2 hours of recirculation. The number of fibrinogen receptors on platelets, which were treated with chymotrypsin after a recirculation, decreased from 41,370 +/- 24,000 to 13,230 +/- 10,230/platelet under the same conditions. The number of antigenic sites for monoclonal antibody reacting with GPIIb/GPIIIa complex of adenosine diphosphate-stimulated platelets decreased from 34,200 +/- 5,940 to 19,500 +/- 9,680/platelet after recirculation. Prostaglandin E1 (0. 3 mumol/L) in the perfusion circuit preserved the ability of platelets to react with fibrinogen. In conclusion, the loss of fibrinogen receptors from the surface of platelet membranes results from the interaction of platelets with the surfaces of perfusion circuits

  12. Use of an anti-platelet monoclonal antibody F (ab')2 fragment for imaging thrombus

    International Nuclear Information System (INIS)

    Loutfi, I.; Stuttle, A.W.J.; Peters, A.M.; George, P.; Lavender, J.P.; Lumley, P.

    1990-01-01

    Ten patients with suspected thrombus have been studied using 111 In-labelled F (ab')2 fragments of P256, a monoclonal antibody which recognizes an epitope on the platelet membrane glycoprotein IIb/IIIa complex. The F (ab')2 fragment was radiolabelled with 111 In via diethylenetri-aminepentamacetic acid to give a specific activity of up to 190 MBq (5mCi) mg - 1 without impairment of immunoreactivity. In vitro platelet aggregation studies showed that the F (ab')2 fragment caused less platelet aggregation than the whole antibody on a molar ratio and was without significant effect upon the sensitivity of platelets to a range of aggregating agents. Platalets were labelled in ten patients by intravenous injection of approximately 100 μg P256 F (ab')2. Of the ten patients studies, six showed localization of activity consistent with platelet accumulation. Localization was clearly seen when associated with thrombus of the lower limbs (three patients: deep vein thrombosis; one patient: aortofemoral graft), and was apparent although less marked in two other cases, one of aortic aneurysm and one of carotid stenosis. Use of radiolabelled P256 F (ab')2 offers a means of non-invasive detection of thrombus which, from in vitro studies, would appear to have less direct effect of platelet behaviour than the whole antibody. (author). 9 refs. 8 figs. 1 tab

  13. Engineered CHO cells for production of diverse, homogeneous glycoproteins

    DEFF Research Database (Denmark)

    Yang, Zhang; Wang, Shengjun; Halim, Adnan

    2015-01-01

    Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells' generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferase...

  14. Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

    Science.gov (United States)

    Gardner, Thomas J.

    2016-01-01

    SUMMARY The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

  15. Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway.

    Science.gov (United States)

    Gardner, Thomas J; Tortorella, Domenico

    2016-09-01

    The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  16. Chemical Synthesis of Glycoproteins with the Specific Installation of Gradient-Enriched15N-Labeled Amino Acids for Getting Insights into Glycoprotein Behavior.

    Science.gov (United States)

    Minh Hien, Nguyen; Izumi, Masayuki; Sato, Hajime; Okamoto, Ryo; Kajihara, Yasuhiro

    2017-05-11

    Elucidating the effects of oligosaccharides on glycoprotein properties, such as local conformational changes, stability, and dynamics, has still been challenging. In this paper, a novel partial 15 N-labeling method for the amide backbone of a synthetic glycoprotein is proposed. Using solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL), thirteen 15 N-labeled amino acids were inserted at specific positions of the protein backbone, while intentionally varying the enrichment of 15 N atoms. This idea discriminated even the same type of amino acid based on the intensities of 1 H- 15 N HSQC signals, combined with classic homonuclear TOCSY and NOESY methods, thus allowing for understanding the dynamics of the local conformation of a synthetic homogeneous glycoprotein. Results suggested that the attachment of an oligosaccharide of either a bi-antennary complex-type or a high-mannose-type did not disturb protein conformation. However, T 1 values suggested that the oligosaccharide influenced dynamics at the local conformation. Temperature-varied circular dichroism (CD) spectra and T 1 values clearly indicated that oligosaccharides appeared to inhibit protein fluctuation or, in other words, stabilize protein structure. This insight into oligosaccharide behavior suggests some further effects on binding affinity between a glycoprotein and its receptor. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Isolation of glycoproteins from brown algae

    DEFF Research Database (Denmark)

    2015-01-01

    The present invention relates to a novel process for the isolation of unique anti-oxidative glycoproteins from the pH precipitated fractions of enzymatic extracts of brown algae. Two brown seaweeds viz, Fucus serratus and Fucus vesiculosus were hydrolysed by using 3 enzymes viz, Alcalase, Viscozyme...

  18. Involvement of Leishmania donovani major surface glycoprotein ...

    Indian Academy of Sciences (India)

    The major surface glycoprotein gp63 of the kinetoplastid protozoal parasite Leishmania is implicated as a ligand mediating uptake of the parasite into, and survival within, the host macrophage. By expressing gp63 antisense RNA from an episomal vector in L. donovani promastigotes, gp63-deficient transfectants were ...

  19. Ubiquitination of exposed glycoproteins by SCFFBXO27 directs damaged lysosomes for autophagy

    Science.gov (United States)

    Yoshida, Yukiko; Yasuda, Sayaka; Fujita, Toshiharu; Hamasaki, Maho; Murakami, Arisa; Kawawaki, Junko; Iwai, Kazuhiro; Saeki, Yasushi; Yoshimori, Tamotsu; Matsuda, Noriyuki; Tanaka, Keiji

    2017-01-01

    Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy. PMID:28743755

  20. Chemical de-O-glycosylation of glycoproteins for applications in LC-based proteomics.

    Science.gov (United States)

    Hanisch, Franz-Georg

    2011-01-01

    This paper describes a cyclic on-column procedure for the sequential degradation of complex O-glycans on proteins by periodate oxidation of sugars and cleavage of oxidation products by elimination. Glycoproteins are immobilized to alkali-stable, reversed-phase Poros 20 beads, desialylated by treatment with dilute trifluoroacetic acid, and de-O-glycosylated by two degradation cycles before the eluted apoproteins are digested with trypsin for analysis by liquid chromatography electrospray ionization-mass spectrometry. Even complex glycan moieties are removed under mild conditions with only minimal effects on structural integrity of the peptide core by fragmentation, dehydration, or racemization of lysine and arginine residues. The protocol is also applicable on gel-immobilized glycoproteins after 1D or 2D gel electrophoresis. Conversion of O-glycoproteins into their corresponding apoproteins results in facilitated accessibility of tryptic cleavage sites, increases the numbers of peptide fragments, and accordingly enhances protein coverage and identification rates within the subproteome of mucin-type O-glycoproteins. The protocol is suitable for automatization, but due to partial elution from the Poros 20 columns it is not recommended for applications on the glycopeptide level.

  1. Secretion of hepatitis C virus envelope glycoproteins depends on assembly of apolipoprotein B positive lipoproteins.

    Directory of Open Access Journals (Sweden)

    Vinca Icard

    Full Text Available The density of circulating hepatitis C virus (HCV particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles has been attributed to an association of the virus with apolipoprotein B (apoB positive and triglyceride rich lipoproteins (TRL likely resulting in hybrid lipoproteins known as lipo-viro-particles (LVP containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The nature of the association of HCV particles with lipoproteins remains elusive and the role of apolipoproteins in the synthesis and assembly of the viral particles is unknown. The human intestinal Caco-2 cell line differentiates in vitro into polarized and apoB secreting cells during asymmetric culture on porous filters. By using this cell culture system, cells stably expressing E1 and E2 secreted the glycoproteins into the basal culture medium after one week of differentiation concomitantly with TRL secretion. Secreted glycoproteins were only detected in apoB containing density fractions. The E1-E2 and apoB containing particles were unique complexes bearing the envelope glycoproteins at their surface since apoB could be co-immunoprecipitated with E2-specific antibodies. Envelope protein secretion was reduced by inhibiting the lipidation of apoB with an inhibitor of the microsomal triglyceride transfer protein. HCV glycoproteins were similarly secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. These data indicate that HCV envelope glycoproteins have the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery even in the absence of the other HCV proteins. A model for LVP assembly is proposed.

  2. Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria

    Science.gov (United States)

    Karav, Sercan; Le Parc, Annabelle; Leite Nobrega de Moura Bell, Juliana Maria; Frese, Steven A.; Kirmiz, Nina; Block, David E.; Barile, Daniela

    2016-01-01

    ABSTRACT Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-β-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis (B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacterium animalis subsp. lactis (B. lactis), a species which does not. Conversely, B. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that B. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B. lactis degraded only 11% of neutral and virtually no (milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides

  3. Insights into the trimeric HIV-1 envelope glycoprotein structure.

    Science.gov (United States)

    Ward, Andrew B; Wilson, Ian A

    2015-02-01

    The HIV-1 envelope glycoprotein (Env) trimer is responsible for receptor recognition and viral fusion with CD4(+) T cells, and is the sole target for neutralizing antibodies. Thus, understanding its molecular architecture is of significant interest. However, the Env trimer has proved to be a challenging target for 3D structure determination. Recent electron microscopy (EM) and X-ray structures have at last enabled us to decipher the structural complexity and unique features of the Env trimer, and how it is recognized by an ever-expanding arsenal of potent broadly neutralizing antibodies. We describe our current knowledge of the Env trimer structure in the context of exciting recent developments in the identification and characterization of HIV broadly neutralizing antibodies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Platelet Glycoprotein lb-1X and Malignancy

    Science.gov (United States)

    2010-09-01

    therapy could benefit the breast cancer patient with malignant disease. Body Below we list the 3 Specific Aims from our original submission (blue font...Muller WJ and Pollard JW. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human...08-1-0576 TITLE: Platelet Glycoprotein lb-1X and Malignancy PRINCIPAL INVESTIGATOR: Dr. Jerry Ware

  5. Platelet Glycoprotein lb-1X and Malignancy

    Science.gov (United States)

    2011-09-01

    independent of pregnancy makes this a useful model to study spontaneous metastasis [26]. To complete this aim, we obtained a mouse colony from Dr. Sandra...mice initiates the spontaneous development of a mammary adenocarcinoma by the age of 8- 10 weeks without pregnancy or any other stimuli. To examine if...patient with systemic lupus erythematosus. Am J Hematol 2001; 67:262-67. 20. Arthur JF, Dunkley S and Andrews RK. Platelet glycoprotein VI-related

  6. Characterization of human immunodeficiency virus type 2 envelope glycoproteins: Dimerization of the glycoprotein precursor during processing

    International Nuclear Information System (INIS)

    Rey, M.A.; Krust, B.; Laurent, A.G.; Montagnier, L.; Hovanessian, A.G.

    1989-01-01

    For glycoproteins with apparent molecular weights of 300,000, 140,000, 125,000, and 36,000 (gp300, gp140, gp125, and gp36) were detectable in human immunodeficiency virus type 2 (HIV-2)-infected cells. They have identical isoelectric points, suggesting that gp300 might be a dimeric form of the immature precursor, gp140. The purified gp300 can be dissociated in a slightly acidic buffer to give rise to monomers of 140,000 molecular weight. Such dissociated monomers and the purified gp140 showed identical patterns of polypeptides after partial proteolysis with Staphylococcus aureus V8 protease. Pulse-chase experiments indicated that gp300 is formed after synthesis of gp140 and before the detection of the mature external envelope glycoprotein, gp125. These results were confirmed by using various inhibitors of glycosylation and inhibitors of trimming enzymes. Dimer formation of the envelope glycoprotein precursor was also observed in cells infected with simian immunodeficiency virus (SIV), a virus closely related to HIV-2. On the other hand, the envelope glycoprotein precursor of HIV-1 did not form a dimer during its processing. Therefore, dimer formation seems to be a specific property of HIV-2 and SIV envelope gene expression. Such transient dimerization of the glycoprotein precursor might be required for its efficient transport to the Golgi apparatus and for its processing

  7. Intracoronary Compared to Intravenous Abciximab in Patients with ST Segment Elevation Myocardial Infarction Treated with Primary Percutaneous Coronary Intervention Reduces Mortality, Target Vessel Revascularization and Reinfarction after 1 Year

    DEFF Research Database (Denmark)

    Iversen, Allan Zeeberg; Galatius, Soeren; Abildgaard, Ulrik

    2011-01-01

    Objectives: Administration of the glycoprotein IIb/IIIa inhibitor abciximab to patients with ST segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (pPCI) improves outcome. Data have suggested that an intracoronary (IC) bolus might be superior...... similar with regard to baseline characteristics. Mortality was reduced from 10% in the IV group to 2.7% in the IC group (p = 0.004). TVR and MI were also reduced with IC administration (TVR: 14.1 vs. 7.6%, p = 0.04; MI: 11.8 vs. 5.4%, p = 0.03). Consequently, patients in the IC treatment arm had...

  8. Early clinical outcomes as a function of use of newer oral P2Y12 inhibitors versus clopidogrel in the EUROMAX trial

    DEFF Research Database (Denmark)

    Huber, Kurt; Ducrocq, Gregory; Hamm, Christian W

    2017-01-01

    prehospital bivalirudin with heparin with optional glycoprotein IIb/IIIa inhibitor treatment in patients with ST-segment elevation myocardial infarction triaged to pPCI. Choice of P2Y12 inhibitor was at the investigator's discretion. In a prespecified analysis, we compared event rates with clopidogrel....... Logistic regression was used to adjust for differences in baseline characteristics. Results: Prasugrel or ticagrelor was given as the loading P2Y12 inhibitor in 49% of 2198 patients and as a maintenance therapy in 59%. No differences were observed in rates of acute stent thrombosis for clopidogrel versus...

  9. Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B

    Energy Technology Data Exchange (ETDEWEB)

    Backovic, Marija [Northwestern Univ., Evanston, IL (United States); Longnecker, Richard [Northwestern Univ., Chicago, IL (United States); Jardetzky, Theodore S [Northwestern Univ., Evanston, IL (United States)

    2009-03-16

    Epstein-Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which forms 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has been solved in its pre and postfusion states, shed light on gB residues that may be involved in conformational changes and membrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distributions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.

  10. Analgesic effects of glycoproteins from Panax ginseng root in mice.

    Science.gov (United States)

    Wang, Ying; Chen, Yinghong; Xu, Hong; Luo, Haoming; Jiang, Ruizhi

    2013-07-30

    The root of Panax ginseng C.A. Mey has various beneficial pharmacological effects. The present study aimed to evaluate the analgesic activities of glycoproteins from the root of Panax ginseng C.A. Mey in mice. Glycoproteins were isolated and purified from the root of Panax ginseng C.A. Mey. Physicochemical properties and molecular mass were determined by chemical assay and HPLC. Acetic acid-induced writhing and hot-plate tests were employed to study the analgesic effect of glycoproteins and compared with that of aspirin or morphine. The locomotor activity was tested in mice by using actophometer. Four glycoproteins were obtained. The glycoproteins which protein content was the highest (73.04%) displayed dose-dependent analgesic effect. In writhing test, the glycoproteins significantly inhibited writhes (Pginseng C.A. Mey exhibited significant analgesic activities and the proteins were the active site, providing evidence for its pharmacal use. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  11. Chemical and Chemoenzymatic Synthesis of Glycoproteins for Deciphering Functions

    Science.gov (United States)

    Wang, Lai-Xi; Amin, Mohammed N.

    2014-01-01

    Summary Glycoproteins are an important class of biomolecules involved in a number of biological recognition processes. However, natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ in the structures of the pendent glycans, which are difficult to separate in pure glycoforms. As a result, synthetic homogeneous glycopeptides and glycoproteins have become indispensable probes for detailed structural and functional studies. A number of elegant chemical and biological strategies have been developed for synthetic construction of tailor-made, full-size glycoproteins to address specific biological problems. In this review, we highlight recent advances in chemical and chemoenzymatic synthesis of homogeneous glycoproteins. Selected examples are given to demonstrate the applications of tailor-made, glycan-defined glycoproteins for deciphering glycosylation functions. PMID:24439206

  12. A combination of "thiol-ene" click chemistry and surface initiated atom transfer radical polymerization: Fabrication of boronic acid functionalized magnetic graphene oxide composite for enrichment of glycoproteins.

    Science.gov (United States)

    Su, Jie; He, Xiwen; Chen, Langxing; Zhang, Yukui

    2018-04-01

    An efficient glycoproteins enrichment platform is one of vital preprocessing steps in biomarker research and in particular glycoproteomics. In this work, a well-defined boronic acid functionalized magnetic graphene oxide nanocomposite (Fe 3 O 4 -GO@PAAPBA) was synthesized for the selective enrichment of glycoproteins from complex biological samples via a novel strategy based on the "thiol-ene" click chemistry and surface initiated atom transfer radical polymerization (SI-ATRP). The initiator of ATRP was anchored to the surface of substrate through "thiol-ene" click reaction. The product Fe 3 O 4 -GO@PAAPBA was successfully synthesized in following SI-ATRP. The Fe 3 O 4 -GO@PAAPBA nanocomposite was characterized by transmission electron microscopy (TEM), Fourier transform-infrared spectroscopy (FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), vibrating sample magnetometry (VSM) and thermogravimetric analysis. The adsorption capacity of Fe 3 O 4 -GO@PAAPBA towards ovalbumin (OVA) and transferrin (Trf) is 471mgg -1 and 450mgg -1 , respectively. The nanocomposite also featured good selectivity to glycoproteins in the mixture of glycoproteins and non-glycoproteins at alkaline (pH 9.0) and physiological conditions (pH 7.4). Furthermore, it can be applied to extract glycoproteins directly from egg white samples. These results have indicated that Fe 3 O 4 -GO@PAAPBA was a potential affinity material in glycoprotein analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Kinetic validation of the models for P-glycoprotein ATP hydrolysis and vanadate-induced trapping. Proposal for additional steps.

    Directory of Open Access Journals (Sweden)

    Miguel Ramón Lugo

    Full Text Available P-Glycoprotein, a member of the ATP-binding cassette (ABC superfamily, is a multidrug transporter responsible for cellular efflux of hundreds of structurally unrelated compounds, including natural products, many clinically used drugs and anti-cancer agents. Expression of P-glycoprotein has been linked to multidrug resistance in human cancers. ABC transporters are driven by ATP hydrolysis at their two cytoplasmic nucleotide-binding domains, which interact to form a closed ATP-bound sandwich dimer. Intimate knowledge of the catalytic cycle of these proteins is clearly essential for understanding their mechanism of action. P-Glycoprotein has been proposed to hydrolyse ATP by an alternating mechanism, for which there is substantial experimental evidence, including inhibition of catalytic activity by trapping of ortho-vanadate at one nucleotide-binding domain, and the observation of an asymmetric occluded state. Despite many studies of P-glycoprotein ATPase activity over the past 20 years, no comprehensive kinetic analysis has yet been carried out, and some puzzling features of its behaviour remain unexplained. In this work, we have built several progressively more complex kinetic models, and then carried out simulations and detailed analysis, to test the validity of the proposed reaction pathway employed by P-glycoprotein for ATP hydrolysis. To establish kinetic parameters for the catalytic cycle, we made use of the large amount of published data on ATP hydrolysis by hamster P-glycoprotein, both purified and in membrane vesicles. The proposed kinetic scheme(s include a high affinity priming reaction for binding of the first ATP molecule, and an independent pathway for ADP binding outside the main catalytic cycle. They can reproduce to varying degrees the observed behavior of the protein's ATPase activity and its inhibition by ortho-vanadate. The results provide new insights into the mode of action of P-glycoprotein, and some hypotheses about the

  14. Podoplanin - a small glycoprotein with many faces

    OpenAIRE

    Ugorski, Maciej; Dziegiel, Piotr; Suchanski, Jaroslaw

    2016-01-01

    Podoplanin is a small membrane glycoprotein with a large number of O-glycoside chains and therefore it belongs to mucin-type proteins. It can be found on the surface of many types of normal cells originating from various germ layers. It is present primarily on the endothelium of lymphatic vessels, type I pneumocytes and glomerular podocytes. Increased levels of podoplanin or its neo-expression have been found in numerous types of human carcinomas, but it is especially common in squamous cell ...

  15. Synthesis of Structures Related to Antifreeze Glycoproteins

    OpenAIRE

    Fyrner, Timmy

    2005-01-01

    In this thesis, synthesis of structures related to antifreeze glycoproteins (AFGPs) are presented. Synthetic routes to a protected carbohydrate derivative, 2,3,4,6-tetra-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-2-azido-4,6-di-O-benzyl-β-D-thio-1-galactopyranoside, and a tBu-Ala-Thr-Ala-Fmoc tripeptide, are described. These compounds are meant to be used in the assembly of AFGPs and analogues thereof. A Gal-GlcN disaccharide was synthesized via glycosylation between the donor, bromo-2-O-benzo...

  16. Glycoprotein component of plant cell walls

    International Nuclear Information System (INIS)

    Cooper, J.B.; Chen, J.A.; Varner, J.E.

    1984-01-01

    The primary wall surrounding most dicotyledonous plant cells contains a hydroxyproline-rich glycoprotein (HRGP) component named extensin. A small group of glycopeptides solubilized from isolated cell walls by proteolysis contained a repeated pentapeptide glycosylated by tri- and tetraarabinosides linked to hydroxyproline and, by galactose, linked to serine. Recently, two complementary approaches to this problem have provided results which greatly increase the understanding of wall extensin. In this paper the authors describe what is known about the structure of soluble extensin secreted into the walls of the carrot root cells

  17. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    Science.gov (United States)

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  18. Identification of N-linked glycoproteins in human milk by hydrophilic interaction liquid chromatography and mass spectrometry

    DEFF Research Database (Denmark)

    Picariello, Gianluca; Ferranti, Pasquale; Mamone, Gianfranco

    2008-01-01

    of glycoconjugates, also including glycoproteins. A number of approaches to describe the complexity of human milk proteome have provided only a partial characterization of restricted classes of N-linked glycoproteins. To achieve this objective, profiling N-linked glycoproteins of human milk was performed...... by Hydrophilic Interaction LC (HILIC) and MS analysis. Glycopeptides were selectively enriched from the protein tryptic digest of human milk samples. Oligosaccharide-free peptides obtained by peptide N-glycosidase F (PNGase F) treatment were characterized by a shotgun MS-based approach, allowing......Breastfeeding is now generally recognized as a critical factor in protecting newborns against infections. An important mechanism responsible for the antibacterial and antiviral effects of breast milk is the prevention of pathogen adhesion to host cell membranes mediated by a number...

  19. A kinetic description of antifreeze glycoprotein activity.

    Science.gov (United States)

    Burcham, T S; Osuga, D T; Yeh, Y; Feeney, R E

    1986-05-15

    The antifreeze glycoproteins (AFGP) of polar fish have the ability to depress the freezing temperature of water approximately 500 times the amount expected based on the number of AFGP molecules in solution; yet AFGP solutions have a purely colligative melting point depression. The difference of solution melting and freezing temperatures is the antifreeze activity of AFGP. One characteristic of AFGP activity that requires further examination is the effect of concentration on antifreeze activity, especially whether the activity saturates at high concentrations or the measured activity increases ad infinitum. This study first surveys the activity of the various antifreeze components from both Pagothenia borchgrevinki and the Arg-containing antifreeze glycoprotein from Eleginus gracilis (EgAF). It was found that all AFGP components examined have a plateau in activity at high concentration, but the actual value of the plateau activity differs between the different length AFGP components and between AFGP and EgAF. While the low molecular weight components of both AFGP and EgAF lose activity at deep supercooling, at high concentration activity is restored. The activity data is then shown to fit a reversible kinetic model of AFGP activity, and the coefficients obtained are used to compare the activity differences between AFGP components and between AFGP and EgAF. The model is also shown to describe the activity of the antifreeze protein of the fish Pseudopleuronectes americanus and the thermal hysteresis protein of the insect, Tenebrio molitor.

  20. Annotating Human P-Glycoprotein Bioassay Data.

    Science.gov (United States)

    Zdrazil, Barbara; Pinto, Marta; Vasanthanathan, Poongavanam; Williams, Antony J; Balderud, Linda Zander; Engkvist, Ola; Chichester, Christine; Hersey, Anne; Overington, John P; Ecker, Gerhard F

    2012-08-01

    Huge amounts of small compound bioactivity data have been entering the public domain as a consequence of open innovation initiatives. It is now the time to carefully analyse existing bioassay data and give it a systematic structure. Our study aims to annotate prominent in vitro assays used for the determination of bioactivities of human P-glycoprotein inhibitors and substrates as they are represented in the ChEMBL and TP-search open source databases. Furthermore, the ability of data, determined in different assays, to be combined with each other is explored. As a result of this study, it is suggested that for inhibitors of human P-glycoprotein it is possible to combine data coming from the same assay type, if the cell lines used are also identical and the fluorescent or radiolabeled substrate have overlapping binding sites. In addition, it demonstrates that there is a need for larger chemical diverse datasets that have been measured in a panel of different assays. This would certainly alleviate the search for other inter-correlations between bioactivity data yielded by different assay setups.

  1. Ammonia transport in the kidney by Rhesus glycoproteins

    Science.gov (United States)

    Verlander, Jill W.

    2014-01-01

    Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4+ with a new model in which specific and regulated transport of both NH3 and NH4+ across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first Rhesus glycoprotein recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiological disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport. PMID:24647713

  2. Bioinformatics Analysis of Envelope Glycoprotein E epitopes of ...

    African Journals Online (AJOL)

    The E glycoprotein of dengue virus is responsible for the viral binding to the receptor. The crystal structure of envelope glycoprotein has already been determined. However, where the well-defined Bcell and T-cell epitopes are located is still a question. Because of the large variations among the four dengue genotypes, it is ...

  3. An improved radioimmunoassay for urinary Tamm-Horsfall glycoprotein

    International Nuclear Information System (INIS)

    Dawnay, A.B. St. J.; Thornley, C.; Cattell, W.R.

    1982-01-01

    A rapid specific radioimmunoassay has been used to measure Tamm-Horsfall glycoprotein (TH glycoprotein) in urine, and the method described. The apparent concentration increased with increasing dilution of urine in water, reaching a plateau at 1 in 20. This increase was greater the higher the osmolality and TH glycoprotein concentration and the lower the pH of the original sample. The apparent concentration of TH glycoprotein in neat or diluted urine was not affected by freezing or by storage at 4 0 C or room temperature for at least 2 days. A physiological range for the urinary excretion rate was established as 22-56 mg/24h, (considerably higher than the amount present in serum) based on samples from 29 individuals with normal renal function, as defined by their creatinine clearance. There was no significant correlation between serum concentrations of TH glycoprotein and its urinary excretion rate, nor between urinary excretion rate and creatinine clearance. (author)

  4. PROTECTIVE EFFECT OF PROLACTIN INDUCED PROTEIN ON ZINC α2-GLYCOPROTEIN AGAINST VARIOUS DENATURANTS

    Directory of Open Access Journals (Sweden)

    Md. Imtaiyaz Hassan

    2012-12-01

    Full Text Available Zinc α2-glycoprotein (ZAG and Prolactin induced protein (PIP are considered as important elements for fertility and biomarker for prostate and breast carcinomas. The stabilities of ZAG alone and its naturally occurring complex with PIP were compared. A significant difference in CD signal was recorded for native ZAG and ZAG-PIP complex against pH-, GdnHCl- and temperature-induced denaturation. These finding suggests that PIP plays a protective role for ZAG against several denaturants. PIP contributes to the hydrophobic as well as electrostatic interactions on ZAG for the complex formation. Moreover, the observed changes in far-UV spectra between ZAG and ZAG-PIP complex in the presence of PEG support the hydrophobic nature of the forces governing the formation of complex. This pH dependent study provides evidence that formation of the complex is a natural event required for physiological function.

  5. Pumping of drugs by P-glycoprotein

    DEFF Research Database (Denmark)

    Litman, Thomas; Skovsgaard, Torben; Stein, Wilfred D

    2003-01-01

    The apparent inhibition constant, Kapp, for the blockade of P-glycoprotein (P-gp) by four drugs, verapamil, cyclosporin A, XR9576 (tariquidar), and vinblastine, was measured by studying their ability to inhibit daunorubicin and calcein-AM efflux from four strains of Ehrlich cells with different...... levels of drug resistance and P-gp content. For daunorubicin as a transport substrate, Kapp was independent of [P-gp] for verapamil but increased strictly linearly with [P-gp] for vinblastine, cyclosporin A, and XR9576. A theoretical analysis of the kinetics of drug pumping and its reversal shows...... but rather, in serial, i.e., a drug that is pumped from the cytoplasmic phase has to pass the preemptive route upon leaving the cell. Our results are consistent with the Sauna-Ambudkar two-step model for pumping by P-gp. We suggest that the vinblastine/cyclosporin A/XR9576-binding site accepts daunorubicin...

  6. Raman optical activity of proteins and glycoproteins

    International Nuclear Information System (INIS)

    Smyth, E.

    2000-03-01

    Raman optical activity (ROA), measured in this project as a small difference in the intensity of Raman scattering from chiral molecules in right- and left-circularly polarised incident laser light, offers the potential to provide more information about the structure of biological molecules in aqueous solution than conventional spectroscopic techniques. Chapter one contains a general discussion of the relative merits of different spectroscopic techniques for structure determination of biomolecules, as well as a brief introduction to ROA. In Chapter two a theoretical analysis of ROA is developed, which extends the discussion in chapter one. The spectrometer setup and sample preparation is then discussed in chapter three. Instrument and sample conditions are monitored to ensure that the best results are obtained. As with any experimental project problems occur, which may result in a degradation of the spectra obtained. The cause of these problems was explored and remedied whenever possible. Chapter four introduces a brief account of protein, glycoprotein and carbohydrate structure and function, with a particular emphasis on the structure of proteins. In the remaining chapters experimental ROA results on proteins and glycoproteins, with some carbohydrate samples, from a wide range of sources are examined. For example, in chapter five some β-sheet proteins are examined. Structural features in these proteins are examined in the extended amide III region of their ROA spectra, revealing that ROA is sensitive to the rigidity or flexibility inherent in proteins. Chapter six concentrates on a group of proteins (usually glycoproteins) known as the serine proteinase inhibitors (serpins). Medically, the serpins are one of the most important groups of proteins of current interest, with wide-ranging implications in conditions such as Down's syndrome, Alzheimer's disease, and emphysema with associated cirrhosis of the liver. With favourable samples and conditions ROA may offer the

  7. Regulation of glycoprotein synthesis in yeast by mating pheromones

    International Nuclear Information System (INIS)

    Tanner, W.

    1984-01-01

    In Saccharomyces cerevisiae, glycosylated proteins amount to less than 2% of the cell protein. Two intensively studied examples of yeast glycoproteins are the external cell wall - associated invertase and the vacuolar carboxypeptidase Y. Recently, it was shown that the mating pheromone, alpha factor, specifically and strongly inhibits the synthesis of N-glycosylated proteins in haploid a cells, whereas O-glycosylated proteins are not affected. In this paper, the pathways of glycoprotein biosynthesis are summarized briefly, and evidence is presented that mating pheomones have a regulatory function in glycoprotein synthesis

  8. Terminal Mannose Residues in Seminal Plasma Glycoproteins of Infertile Men Compared to Fertile Donors

    Directory of Open Access Journals (Sweden)

    Beata Olejnik

    2015-07-01

    Full Text Available The impact of seminal plasma components on the fertilization outcomes in humans is still under question. The increasing number of couples facing problems with conception raises the need for predictive biomarkers. Detailed understanding of the molecular mechanisms accompanying fertilization remains another challenge. Carbohydrate–protein recognition may be of key importance in this complex field. In this study, we analyzed the unique glycosylation pattern of seminal plasma proteins, the display of high-mannose and hybrid-type oligosaccharides, by means of their reactivity with mannose-specific Galanthus nivalis lectin. Normozoospermic infertile subjects presented decreased amounts of lectin-reactive glycoepitopes compared to fertile donors and infertile patients with abnormal semen parameters. Glycoproteins containing unveiled mannose were isolated in affinity chromatography, and 17 glycoproteins were identified in liquid chromatography-tandem mass spectrometry with electrospray ionization. The N-glycome of the isolated glycoproteins was examined in matrix-assisted laser desorption ionization mass spectrometry. Eleven out of 27 identified oligosaccharides expressed terminal mannose residues, responsible for lectin binding. We suggest that lowered content of high-mannose and hybrid type glycans in normozoospermic infertile patients may be associated with impaired sperm protection from preterm capacitation and should be considered in the search for new infertility markers.

  9. A Novel Fiber Optic Surface Plasmon Resonance Biosensors with Special Boronic Acid Derivative to Detect Glycoprotein

    Directory of Open Access Journals (Sweden)

    Yang Zhang

    2017-10-01

    Full Text Available We proposed and demonstrated a novel tilted fiber Bragg grating (TFBG-based surface plasmon resonance (SPR label-free biosensor via a special boronic acid derivative to detect glycoprotein with high sensitivity and selectivity. TFBG, as an effective sensing element for optical sensing in near-infrared wavelengths, possess the unique capability of easily exciting the SPR effect on fiber surface which coated with a nano-scale metal layer. SPR properties can be accurately detected by measuring the variation of transmitted spectra at optical communication wavelengths. In our experiment, a 10° TFBG coated with a 50 nm gold film was manufactured to stimulate SPR on a sensor surface. To detect glycoprotein selectively, the sensor was immobilized using designed phenylboronic acid as the recognition molecule, which can covalently bond with 1,2- or 1,3-diols to form five- or six-membered cyclic complexes for attaching diol-containing biomolecules and proteins. The phenylboronic acid was synthetized with long alkyl groups offering more flexible space, which was able to improve the capability of binding glycoprotein. The proposed TFBG-SPR sensors exhibit good selectivity and repeatability with a protein concentration sensitivity up to 2.867 dB/ (mg/mL and a limit of detection (LOD of 15.56 nM.

  10. A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity.

    Science.gov (United States)

    York, Joanne; Nunberg, Jack H

    2018-01-01

    For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

  11. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Beta-2-glycoprotein I immunological test system....5430 Beta-2-glycoprotein I immunological test system. (a) Identification. A beta-2-glycoprotein I... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of beta-2...

  12. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Beta-2-glycoprotein III immunological test system....5440 Beta-2-glycoprotein III immunological test system. (a) Identification. A beta-2-glycoprotein III... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of beta-2...

  13. Mechanisms involved in the antiplatelet effect of C-phycocyanin.

    Science.gov (United States)

    Chiu, Hui-Fen; Yang, Shih-Ping; Kuo, Yu-Ling; Lai, Yuan-Shu; Chou, Tz-Chong

    2006-02-01

    C-phycocyanin (cpc), a biliprotein isolated from Spirulina platensis, has been reported to exert many therapeutic and nutritional values. In the present study, we examined whether cpc has an antiplatelet activity in vitro and further investigated the possible anti-aggregatory mechanisms involved. Our results showed that preincubation of cpc (1-50 microg/ml) with rabbit washed platelets dose-dependently inhibited the platelet aggregation induced by collagen (10 microg/ml) or arachidonic acid (100 microm), with an IC50 of about 10 microg/ml. Furthermore, the thromboxane B2 formation caused by collagen or arachidonic acid was significantly inhibited by cpc due to suppression of cyclooxygenase and thromboxane synthase activity. Similarly, the rise of platelet intracellular calcium level stimulated by arachidonic acid and collagen-induced platelet membrane surface glycoprotein IIb/IIIa expression were also attenuated by cpc. In addition, cpc itself significantly increased the platelet membrane fluidity and the cyclic AMP level through inhibiting cyclic AMP phosphodiesterase activity. These findings strongly demonstrate that cpc is an inhibitor of platelet aggregation, which may be associated with mechanisms including inhibition of thromboxane A2 formation, intracellular calcium mobilization and platelet surface glycoprotein IIb/IIIa expression accompanied by increasing cyclic AMP formation and platelet membrane fluidity.

  14. Novel agents for anti-platelet therapy

    Directory of Open Access Journals (Sweden)

    Ji Xuebin

    2011-11-01

    Full Text Available Abstract Anti-platelet therapy plays an important role in the treatment of patients with thrombotic diseases. The most commonly used anti-platelet drugs, namely, aspirin, ticlopidine, and clopidogrel, are effective in the prevention and treatment of cardio-cerebrovascular diseases. Glycoprotein IIb/IIIa antagonists (e.g., abciximab, eptifibatide and tirofiban have demonstrated good clinical benefits and safety profiles in decreasing ischemic events in acute coronary syndrome. However, adverse events related to thrombosis or bleeding have been reported in cases of therapy with glycoprotein IIb/IIIa antagonists. Cilostazol is an anti-platelet agent used in the treatment of patients with peripheral ischemia, such as intermittent claudication. Presently, platelet adenosine diphosphate P2Y(12 receptor antagonists (e.g., clopidogrel, prasugrel, cangrelor, and ticagrelor are being used in clinical settings for their pronounced protective effects. The new protease-activated receptor antagonists, vorapaxar and atopaxar, potentially decrease the risk of ischemic events without significantly increasing the rate of bleeding. Some other new anti-platelet drugs undergoing clinical trials have also been introduced. Indeed, the number of new anti-platelet drugs is increasing. Consequently, the efficacy of these anti-platelet agents in actual patients warrants scrutiny, especially in terms of the hemorrhagic risks. Hopefully, new selective platelet inhibitors with high anti-thrombotic efficiencies and low hemorrhagic side effects can be developed.

  15. Expression of glycoprotein VI in vascular endothelial cells.

    Science.gov (United States)

    Sun, Bing; Tao, Lian; Lin, Shihua; Calingasan, Noel Y; Li, Jess; Tandon, Narendra N; Yoshitake, Masuhiro; Kambayashi, Jun-ichi

    2003-06-01

    Glycoprotein (GP) VI, a collagen receptor, plays a important role in collagen-mediated platelet aggregation and adhesion. To date, GPVI expression has been found only in platelets and megakaryocytes. In the present studies, we have demonstrated that GPVI was also expressed in cultured human umbilical vein endothelial cells (HUVEC) at both transcript and protein levels. Using a GPVI-specific probe, a approximately 6-kb band was detected in HUVEC as well as in platelets and megakaryoblastic cell lines by Northern blotting. Using polyclonal antibodies raised against platelet GPVI peptides, the same size band (57 kDa) was labeled with convulxin (CVX) after immuo-precipitation in both HUVEC and platelet lysates. In addition, a approximately 70-kDa band was also labeled in HUVEC. Surface expression of GPVI in HUVEC was confirmed by flow cytometry with GPVI-specific IgG or by direct labeling with FITC-conjugated CVX. Since HUVEC lack FcRgamma chain that forms complex with GPVI in platelets for signaling process, the function of GPVI in vascular endothelial cells remains to be determined.

  16. Infiltração cutânea na leucemia megacariocítica aguda com expressão de CD56 Cutaneous infiltration in acute megakaryocytic leukemia with CD56 expression

    Directory of Open Access Journals (Sweden)

    Mariela G. Farias

    2007-06-01

    Full Text Available A LMA-M7 é um subtipo raro de leucemia mielóide aguda (LMA. Está freqüentemente associada a mielofibrose e representa um subtipo de mau prognóstico. Raramente apresenta infiltração em sítios extramedulares. O aspirado de medula óssea ou biópsia mostra uma população de células pleomórficas e basofílicas, que podem apresentar projeções citoplasmáticas. A utilização da imunofenotipagem é essencial para o diagnóstico de LMA-M7. O imunofenótipo característico apresenta uma população de células leucêmicas com ausência da maioria dos marcadores linfóides e mielóides de superfície, porém com expressão para os antígenos da linhagem megacariocítica: CD41a (complexo glicoprotéico IIb/IIIa, CD42b (glicoproteína Ib e/ou CD61 (glicoproteína IIIa, ou antígeno relacionado ao fator VIII. Freqüentemente, a coloração citoquímica Sudan Black para os blastos megacariocíticos é negativa; neste caso, foi positiva para 40% das células analisadas. A presença de CD56, cuja expressão aberrante em algumas leucemias mielóides é indicativo de mau prognóstico, pode estar associada à infiltração da pele.AML-M7 is a rare subtype of acute myeloid leukemia (AML. It is frequently associated with myelofibrosis and corresponds to a poor prognosis subtype. It rarely presents with infiltration at extramedullary sites. The bone marrow aspirate or biopsy identifies pleomorphic and basophilic cell populations that may present with cytoplasmatic projections. The use of immunophenotyping is essential for the diagnosis of AML-M7. The characteristic immunophenotype presents a leukemic cell population without most lymphoid and myeloid surface markers, but with an expression of the megakaryocytic antigens: CD41a (glycoprotein complex IIb/IIIa, CD42b (glycoprotein Ib and/or CD61 (glycoprotein IIIa, or the factor VIII-related antigen. The cytochemical stain Sudan Black is frequently negative for megakaryocytic blasts; in this case, it was

  17. Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

    Directory of Open Access Journals (Sweden)

    Daniel L Glauser

    Full Text Available Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4 entry machinery--gB, gH/gL and gp150--changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion.

  18. Mass spectrometry-based proteomics of fungal wall glycoproteins

    NARCIS (Netherlands)

    Yin, Q.Y.; de Groot, P.W.J.; de Koster, C.G.; Klis, F.M.

    2008-01-01

    The manifold functions of fungal wall glycoproteins include maintenance of cell wall integrity, homotypic and heterotypic adhesion, biofilm formation, acquisition of iron and sterols, protein degradation and coping with oxidative stress. Transcriptome studies indicate that the expression levels of

  19. Detection of glycoproteins in the Acanthamoeba plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Paatero, G.I.L. (Abo Akademi (Finland)); Gahmberg, C.G. (Univ. of Helsinki (Finland))

    1988-11-01

    In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by {sup 125}I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB{sup 3}H{sub 4} and galactose oxidase/NaB{sup 3}H{sub 4} labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M{sub r} of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with ({sup 35}S)methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.

  20. Detection of glycoproteins in the Acanthamoeba plasma membrane

    International Nuclear Information System (INIS)

    Paatero, G.I.L.; Gahmberg, C.G.

    1988-01-01

    In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by 125 I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB 3 H 4 and galactose oxidase/NaB 3 H 4 labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M r of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with [ 35 S]methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis

  1. Systemic alteration of cell-surface and secreted glycoprotein expression in malignant breast cancer cell lines

    OpenAIRE

    Timpe, Leslie C; Yen, Roger; Haste, Nicole V; Litsakos-Cheung, Christina; Yen, Ten-Yang; Macher, Bruce A

    2013-01-01

    Breast cancer cell lines express fewer transmembrane and secreted glycoproteins than nonmalignant ones. The objective of these experiments was to characterize the changes in the expression of several hundred glycoproteins quantitatively. Secreted and cell-surface glycoproteins were isolated using a glycoprotein capture protocol and then identified by tandem mass spectrometry. Glycoproteins expressed by a group of cell lines originating from malignant tumors of the breast were compared with th...

  2. Enzymatic sulfation of mucus glycoprotein in gastric mucosa

    International Nuclear Information System (INIS)

    Liau, Y.H.; Carter, S.R.; Gwozdzinski, K.; Nadziejko, C.; Slomiany, A.; Slomiany, B.L.

    1986-01-01

    Among the posttranslational modifications that mucus glycoprotein undergo prior to secretion into the gastric lumen is the process of sulfation of the carbohydrate chains. These sulfate groups impart strongly negative charge to nucus glycoprotein and are thought to play a major role in the maintenance of gastric mucosal integrity. The authors report here the presence and some properties of an enzyme involved in the sulfation of gastric mucus glycoprotein. The sulfotransferase activity which catalyzes the transfer of sulfate ester group from PAPS to mucus glycoprotein was located in the detergent extracts of the microsomal fraction of rat gastric mucosa. Optimum enzymatic activity for sulfation of gastric mucin was obtained using 0.5% Triton X-100 and 25mM NaF at a pH of 6.8. ATP, ADP, MgCl 2 and MnCl 2 at concentrations examined were inhibitory. Under optimal conditions, the rate of sulfate incorporation was proportional to the microsomal enzyme protein concentration up to 50μg and remained constant with time of incubation for at least 1h. The apparent Km value of the enzyme for gastric mucus glycoprotein was 8.3 x 10 -6 M. The 35 S-labeled product of the enzyme reaction cochromatographed on Bio-Gel A-50 with gastric mucin, and gave on CsCl equilibrium density gradient centrifugation a band at the density of 1.48 in which the 35 S label coincided with the glycoprotein

  3. Nucleic acid-binding glycoproteins which solubilize nucleic acids in dilute acid: re-examination of the Ustilago maydis glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Unrau, P.; Champ, D.R.; Young, J.L.; Grant, C.E.

    1980-01-01

    Holloman reported the isolation from Ustilago maydis of a glycoprotein which prevented the precipitation of nucleic acids in cold 5% trichloroacetic acid. Two glycoprotein fractions from U. maydis with this nucleic acid-solubilizing activity were isolated in our laboratory using improved purification procedures. The activity was not due to nuclease contamination. The glycoproteins are distinguished by: their ability to bind to concanavalin A-Sepharose; their differential binding to double- and single-stranded deoxyribonucleic acid, and to ribonucleic acid; their molecular weights (46,000 and 69,000); and the relative amounts present in growing versus nongrowing cells. Both fractions required sulfhydryl-reducing conditions for optimal yields, specific activity, and stability. Nucleic acid binding was cooperative, the minimum number of glycoproteins required to make a native T7 DNA molecule soluble in dilute acid being estimated at 2 and 15, respectively.

  4. Alternative promoter usage of the membrane glycoprotein CD36

    Directory of Open Access Journals (Sweden)

    Whatling Carl

    2006-03-01

    Full Text Available Abstract Background CD36 is a membrane glycoprotein involved in a variety of cellular processes such as lipid transport, immune regulation, hemostasis, adhesion, angiogenesis and atherosclerosis. It is expressed in many tissues and cell types, with a tissue specific expression pattern that is a result of a complex regulation for which the molecular mechanisms are not yet fully understood. There are several alternative mRNA isoforms described for the gene. We have investigated the expression patterns of five alternative first exons of the CD36 gene in several human tissues and cell types, to better understand the molecular details behind its regulation. Results We have identified one novel alternative first exon of the CD36 gene, and confirmed the expression of four previously known alternative first exons of the gene. The alternative transcripts are all expressed in more than one human tissue and their expression patterns vary highly in skeletal muscle, heart, liver, adipose tissue, placenta, spinal cord, cerebrum and monocytes. All alternative first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins. The alternative promoters lack TATA-boxes and CpG islands. The upstream region of exon 1b contains several features common for house keeping gene and monocyte specific gene promoters. Conclusion Tissue-specific expression patterns of the alternative first exons of CD36 suggest that the alternative first exons of the gene are regulated individually and tissue specifically. At the same time, the fact that all first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins may suggest that the alternative first exons are coregulated in this cell type and environmental condition. The molecular mechanisms regulating CD36 thus appear to be unusually complex, which might reflect the multifunctional role of the gene in different tissues and cellular conditions.

  5. Antifreeze glycoprotein agents: structural requirements for activity.

    Science.gov (United States)

    Carvajal-Rondanelli, Patricio A; Marshall, Sergio H; Guzman, Fanny

    2011-11-01

    Antifreeze glycoproteins (AFGPs) are considered to be the most efficient means to reduce ice damage to cell tissues since they are able to inhibit growth and crystallization of ice. The key element of antifreeze proteins is to act in a non-colligative manner which allows them to function at concentrations 300-500 times lowers than other dissolved solutes. During the past decade, AFGPs have demonstrated tremendous potential for many pharmaceutical and food applications. Presently, the only route to obtain AFGPs involves the time consuming and expensive process of isolation and purification from deep-sea polar fishes. Unfortunately, it is not amenable to mass production and commercial applications. The lack of understanding of the mechanism through which the AFGPs inhibit ice growth has also hampered the realization of industrial and biotechnological applications. Here we report the structural motifs that are essential for antifreeze activity of AFGPs, and propose a unified mechanism based on both recent studies of short alanine peptides and structure activity relationship of synthesized AFGPs. Copyright © 2011 Society of Chemical Industry.

  6. P-glycoprotein targeted nanoscale drug carriers

    KAUST Repository

    Li, Wengang

    2013-02-01

    Multi-drug resistance (MDR) is a trend whereby tumor cells exposed to one cytotoxic agent develop cross-resistance to a range of structurally and functionally unrelated compounds. P -glycoprotein (P -gp) efflux pump is one of the mostly studied drug carrying processes that shuttle the drugs out of tumor cells. Thus, P -gp inhibitors have attracted a lot of attention as they can stop cancer drugs from being pumped out of target cells with the consumption of ATP. Using quantitive structure activity relationship (QSAR), we have successfully synthesized a series of novel P -gp inhibitors. The obtained dihydropyrroloquinoxalines series were fully characterized and then tested against bacterial and tumor assays with over-expressed P -gps. All compounds were bioactive especially compound 1c that had enhanced antibacterial activity. Furthermore, these compounds were utilized as targeting vectors to direct drug delivery vehicles such as silica nanoparticles (SNPs) to cancerous Hela cells with over expressed P -gps. Cell uptake studies showed a successful accumulation of these decorated SNPs in tumor cells compared to undecorated SNPs. The results obtained show that dihydropyrroloquinoxalines constitute a promising drug candidate for targeting cancers with MDR. Copyright © 2013 American Scientific Publishers All rights reserved.

  7. Cellular and biophysical evidence for interactions between adenosine triphosphate and P-glycoprotein substrates

    DEFF Research Database (Denmark)

    Abraham, E H; Shrivastav, B; Salikhova, A Y

    2001-01-01

    P-glycoprotein is involved with the removal of drugs, most of them cations, from the plasma membrane and cytoplasm. Pgp is also associated with movement of ATP, an anion, from the cytoplasm to the extracellular space. The central question of this study is whether drug and ATP transport associated...... provides a framework for understanding the role of erythrocytes in drug resistance. The erythrocyte consists of a membrane surrounding a millimolar pool of ATP. Mammalian RBCs have no nucleus or DNA drug/toxin targets. From the perspective of drug/ATP complex formation, the RBC serves as an important...

  8. P-glycoprotein activity and biological response

    International Nuclear Information System (INIS)

    Vaalburg, W.; Hendrikse, N.H.; Elsinga, P.H.; Bart, J.; Waarde, A. van

    2005-01-01

    P-glycoprotein (P-gp) is a transmembrane drug efflux pump encoded by the MDR-1 gene in humans. Most likely P-gp protects organs against endogenous and exogenous toxins by extruding toxic compounds such as chemotherapeutics and other drugs. Many drugs are substrates for P-gp. Since P-gp is also expressed in the blood-brain barrier, P-gp substrates reach lower concentrations in the brain than in P-gp-negative tissues. Failure of response to chemotherapy of malignancies can be due to intrinsic or acquired drug resistance. Many tumors are multidrug resistant (MDR); resistant to several structurally unrelated chemotherapeutic agents. Several mechanisms are involved in MDR of which P-gp is studied most extensively. P-gp extrudes drugs out of tumor cells resulting in decreased intracellular drug concentrations, leading to the MDR phenotype. Furthermore, the MDR-1 gene exhibits several single nucleotide polymorphisms, some of which result in different transport capabilities. P-gp functionality and the effect of P-gp modulation on the pharmacokinetics of novel and established drugs can be studied in vivo by positron emission tomography (PET) using carbon-11 and fluorine-18-labeled P-gp substrates and modulators. PET may demonstrate the consequences of genetic differences on tissue pharmacokinetics. Inhibitors such as calcium-channel blockers (verapamil), cyclosporin A, ONT-093, and XR9576 can modulate the P-gp functionality. With PET the effect of P-gp modulation on the bioavailability of drugs can be investigated in humans in vivo. PET also allows the measurement of the efficacy of newly developed P-gp modulators

  9. Physical Properties of the Glycoprotein Mucin

    Science.gov (United States)

    Matthews, Garrett; Davis, William; Superfine, Richard; Boucher, Richard

    2003-03-01

    Epithelial cell surfaces are covered by a protective gel known as mucus. The physiological function of this gel depends on its rheological properties, and these properties are largely derived from the secreted glycoprotein mucin. The genetic disease Cystic Fibrosis (CF) is characterized by the adhesion of thick, viscous mucus on these tissues. In the lungs, this results in the interruption of mucus transport thus compromising the first line of defense against pathogens in these tissues. In order to restore the flow of tracheobronchial mucus out of the body, knowledge of the molecular and physical properties of mucin and mucin solutions would be greatly beneficial. The present model for these molecules is that of a long linear strand consisting of highly glycosylated regions linked by cystein-rich globular regions. It is thought that the globular regions may interact either through intermolecular disulfide bonds or through hydrophobic interactions. It has also been speculated that the glycosylated regions may have lectin-like interactions. In the present work, single mucin molecules were imaged at high resolution using atomic force microscopy (AFM). Phase mode imaging was used to map the interactions between functionalized AFM tips and the molecular topography. Additionally, using force-distance curves with the AFM, the adhesion between mucin bound tips and cell surface glycocalyx and glycocalyx-like model surfaces, was measured. And, finally, the viscoelastic properties of mucin solutions were measured using the recently developed technique, single particle tracking microrheology. A model is being developed that will incorporate the properties of mucins beginning at the single molecule and ending with the bulk viscoelastic properties.

  10. Removal of N-Linked Glycosylations at Acidic pH by PNGase A Facilitates Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of N-Linked Glycoproteins

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Comamala Grau, Gerard; Trelle, Morten Beck

    2016-01-01

    and their glycans challenging for most analytical techniques. Hydrogen/deuterium exchange monitored by mass spectrometry is a sensitive technique for investigation of protein conformational dynamics of complex heterogeneous proteins in solution. N-linked glycoproteins however pose a challenge for HDX-MS. HDX...

  11. Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

    International Nuclear Information System (INIS)

    Peerschke, E.I.

    1991-01-01

    Previous studies indicated a correlation between the formation of EDTA-resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA-resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA-resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding

  12. Structures and Functions of Pestivirus Glycoproteins: Not Simply Surface Matters.

    Science.gov (United States)

    Wang, Fun-In; Deng, Ming-Chung; Huang, Yu-Liang; Chang, Chia-Yi

    2015-06-29

    Pestiviruses, which include economically important animal pathogens such as bovine viral diarrhea virus and classical swine fever virus, possess three envelope glycoproteins, namely Erns, E1, and E2. This article discusses the structures and functions of these glycoproteins and their effects on viral pathogenicity in cells in culture and in animal hosts. E2 is the most important structural protein as it interacts with cell surface receptors that determine cell tropism and induces neutralizing antibody and cytotoxic T-lymphocyte responses. All three glycoproteins are involved in virus attachment and entry into target cells. E1-E2 heterodimers are essential for viral entry and infectivity. Erns is unique because it possesses intrinsic ribonuclease (RNase) activity that can inhibit the production of type I interferons and assist in the development of persistent infections. These glycoproteins are localized to the virion surface; however, variations in amino acids and antigenic structures, disulfide bond formation, glycosylation, and RNase activity can ultimately affect the virulence of pestiviruses in animals. Along with mutations that are driven by selection pressure, antigenic differences in glycoproteins influence the efficacy of vaccines and determine the appropriateness of the vaccines that are currently being used in the field.

  13. Structures and Functions of Pestivirus Glycoproteins: Not Simply Surface Matters

    Directory of Open Access Journals (Sweden)

    Fun-In Wang

    2015-06-01

    Full Text Available Pestiviruses, which include economically important animal pathogens such as bovine viral diarrhea virus and classical swine fever virus, possess three envelope glycoproteins, namely Erns, E1, and E2. This article discusses the structures and functions of these glycoproteins and their effects on viral pathogenicity in cells in culture and in animal hosts. E2 is the most important structural protein as it interacts with cell surface receptors that determine cell tropism and induces neutralizing antibody and cytotoxic T-lymphocyte responses. All three glycoproteins are involved in virus attachment and entry into target cells. E1-E2 heterodimers are essential for viral entry and infectivity. Erns is unique because it possesses intrinsic ribonuclease (RNase activity that can inhibit the production of type I interferons and assist in the development of persistent infections. These glycoproteins are localized to the virion surface; however, variations in amino acids and antigenic structures, disulfide bond formation, glycosylation, and RNase activity can ultimately affect the virulence of pestiviruses in animals. Along with mutations that are driven by selection pressure, antigenic differences in glycoproteins influence the efficacy of vaccines and determine the appropriateness of the vaccines that are currently being used in the field.

  14. An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor

    International Nuclear Information System (INIS)

    Mische, Claudia C.; Yuan Wen; Strack, Bettina; Craig, Stewart; Farzan, Michael; Sodroski, Joseph

    2005-01-01

    The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate

  15. Providing a molecular mechanism for P-glycoprotein; why would I bother?

    Science.gov (United States)

    Callaghan, Richard

    2015-10-01

    It is almost 40 years since the drug efflux pump P-glycoprotein (permeability glycoprotein or P-gp) was shown to confer multi-drug resistance in cancer cells. This protein has been one of the most extensively investigated transport proteins due to its intriguing mechanism and its affect in oncology. P-gp is known to interact with over 300 compounds and the ability to achieve this has not yet been revealed. Following the binding of substrate and nucleotide, a complex series of conformational changes in the membrane and cytosolic domains translocates substrate across the membrane. Despite over 30 years of biochemical investigation, the availability of structural data and a plethora of chemical tools to modulate its function, the molecular mechanism remains a mystery. In addition, overcoming its activity in resistant cancer cells has not been achieved in the clinic, thereby garnering some degree of pessimism in the field. This review highlights the progress that has been achieved in understanding this complex protein and the value of undertaking molecular studies. © 2015 Authors; published by Portland Press Limited.

  16. Multiple genes encode the major surface glycoprotein of Pneumocystis carinii

    DEFF Research Database (Denmark)

    Kovacs, J A; Powell, F; Edman, J C

    1993-01-01

    hydrophobic region at the carboxyl terminus. The presence of multiple related msg genes encoding the major surface glycoprotein of P. carinii suggests that antigenic variation is a possible mechanism for evading host defenses. Further characterization of this family of genes should allow the development......The major surface antigen of Pneumocystis carinii, a life-threatening opportunistic pathogen in human immunodeficiency virus-infected patients, is an abundant glycoprotein that functions in host-organism interactions. A monoclonal antibody to this antigen is protective in animals, and thus...... blot studies using chromosomal or restricted DNA, the major surface glycoproteins are the products of a multicopy family of genes. The predicted protein has an M(r) of approximately 123,000, is relatively rich in cysteine residues (5.5%) that are very strongly conserved, and contains a well conserved...

  17. Australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing

    International Nuclear Information System (INIS)

    Tropea, J.E.; Molyneux, R.J.; Kaushal, G.P.; Pan, Y.T.; Mitchell, M.; Elbein, A.D.

    1989-01-01

    Australine is a polyhydroxylated pyrrolizidine alkaloid that was isolated from the seeds of the Australian tree Castanospermum australe and characterized by NMR and X-ray diffraction analysis. Since swainsonine and catanospermine are polyhydroxylated indolizidine alkaloids that inhibit specific glycosidases, the authors tested australine against a variety of exoglycosidases to determine whether it would inhibit any of these enzymes. This alkaloid proved to be a good inhibitor of the α-glucosidase amyloglucosidase (50% inhibition at 5.8 μM), but it did not inhibit β-glucosidase, α- or β-mannosidase, or α- or β-galactosidase. The inhibition of amyloglucosidase was of a competitive nature. Australine also inhibited the glycoprotein processing enzyme glucosidase I, but had only slight activity toward glucosidase II. When incubated with cultured cells, this alkaloid inhibited glycoprotein processing at the glucosidase I step and caused the accumulation of glycoproteins with Glc 3 Man 7-9 (GlcNAc) 2 -oligosaccharides

  18. Low-molecular-weight heparin therapy in percutaneous coronary intervention: the NICE 1 and NICE 4 trials. National Investigators Collaborating on Enoxaparin Investigators.

    Science.gov (United States)

    Young, J J; Kereiakes, D J; Grines, C L

    2000-12-01

    Early coronary intervention in patients with non-ST segment elevation acute coronary syndromes (ACS) may be facilitated by adjunctive pharmacotherapies. Concomitant therapies such as low-molecular-weight heparins and platelet glycoprotein (GP) IIb/IIIa receptor blockade offer advantages in safety and efficacy during coronary intervention. Data from randomized clinical trials support the administration of both enoxaparin and platelet GP IIb/IIIa blockade to patients who present with non-ST segment elevation ACS. Enoxaparin, with its proven efficacy, predictability of action, and ease of administration, has been shown to be superior to unfractionated heparin in preventing major coronary events. Abciximab administration during percutaneous coronary intervention (PCI) reduces the incidence of ischemic adverse outcomes and appears to improve survival in long-term follow-up. The preliminary experience with combining these two therapies during PCI in the NICE 4 trial demonstrates a low incidence of minor/major bleeding and transfusion, and infrequent major cardiac events to 30 days follow-up. Algorithms for the use of these newer adjunctive pharmacotherapies in the care of patients presenting to the cardiac catheterization laboratory are presented.

  19. Multiple genes encode the major surface glycoprotein of Pneumocystis carinii

    DEFF Research Database (Denmark)

    Kovacs, J A; Powell, F; Edman, J C

    1993-01-01

    this antigen is a good candidate for development as a vaccine to prevent or control P. carinii infection. We have cloned and sequenced seven related but unique genes encoding the major surface glycoprotein of rat P. carinii. Partial amino acid sequencing confirmed the identity of these genes. Based on Southern...... blot studies using chromosomal or restricted DNA, the major surface glycoproteins are the products of a multicopy family of genes. The predicted protein has an M(r) of approximately 123,000, is relatively rich in cysteine residues (5.5%) that are very strongly conserved, and contains a well conserved...

  20. A Functional Henipavirus Envelope Glycoprotein Pseudotyped Lentivirus Assay System

    Directory of Open Access Journals (Sweden)

    Broder Christopher C

    2010-11-01

    Full Text Available Abstract Background Hendra virus (HeV and Nipah virus (NiV are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4 containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP gene encoding human immunodeficiency virus type-1 (HIV-1 genome in conjunction with the HeV and NiV fusion (F and attachment (G glycoproteins. Results Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2 peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F

  1. Intestinal mucus and juice glycoproteins have a liquid crystalline structure

    International Nuclear Information System (INIS)

    Denisova, E.A.; Lazarev, P.I.; Vazina, A.A.; Zheleznaya, L.A.

    1985-01-01

    X-ray diffraction patterns have been obtained from the following components of canine gastrointestinal tract: (1) native small intestine mucus layer; (2) the precipitate of the flocks formed in the duodenal juice with decreasing pH; (3) concentrated solutions of glycoproteins isolated from the duodenal juice. The X-ray patterns consist of a large number of sharp reflections of spacings between about 100 and 4 A. Some reflections are common for all components studied. All the patterns are interpreted as arising from the glycoprotein molecules ordered into a liquid crystalline structure. (author)

  2. Multiple genes encode the major surface glycoprotein of Pneumocystis carinii

    DEFF Research Database (Denmark)

    Kovacs, J A; Powell, F; Edman, J C

    1993-01-01

    The major surface antigen of Pneumocystis carinii, a life-threatening opportunistic pathogen in human immunodeficiency virus-infected patients, is an abundant glycoprotein that functions in host-organism interactions. A monoclonal antibody to this antigen is protective in animals, and thus...... hydrophobic region at the carboxyl terminus. The presence of multiple related msg genes encoding the major surface glycoprotein of P. carinii suggests that antigenic variation is a possible mechanism for evading host defenses. Further characterization of this family of genes should allow the development...... of novel approaches to the control of this pathogen....

  3. Dimeric architecture of the Hendra virus attachment glycoprotein: evidence for a conserved mode of assembly.

    Science.gov (United States)

    Bowden, Thomas A; Crispin, Max; Harvey, David J; Jones, E Yvonne; Stuart, David I

    2010-06-01

    Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed beta-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to alpha-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60 degrees ) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses.

  4. Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host.

    Science.gov (United States)

    Ravidà, Alessandra; Cwiklinski, Krystyna; Aldridge, Allison M; Clarke, Paul; Thompson, Roisin; Gerlach, Jared Q; Kilcoyne, Michelle; Hokke, Cornelis H; Dalton, John P; O'Neill, Sandra M

    2016-10-01

    Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host*

    Science.gov (United States)

    Ravidà, Alessandra; Cwiklinski, Krystyna; Aldridge, Allison M.; Clarke, Paul; Thompson, Roisin; Gerlach, Jared Q.; Kilcoyne, Michelle; Hokke, Cornelis H.; Dalton, John P.; O'Neill, Sandra M.

    2016-01-01

    Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. PMID:27466253

  6. P-glycoprotein ABCB1: a major player in drug handling by mammals

    NARCIS (Netherlands)

    Borst, Piet; Schinkel, Alfred H.

    2013-01-01

    Mammalian P-glycoproteins are active drug efflux transporters located in the plasma membrane. In the early nineties, we generated knockouts of the three P-glycoprotein genes of mice, the Mdr1a, Mdr1b, and Mdr2 P-glycoproteins, now known as Abcb1a, Abcb1b, and Abcb4, respectively. In the JCI papers

  7. Cereal n-glycoproteins enrichment by lectin affinity monolithic chromatography

    Czech Academy of Sciences Publication Activity Database

    Flodrová, Dana; Bobálová, Janette; Laštovičková, Markéta

    2016-01-01

    Roč. 44, č. 2 (2016), s. 286-297 ISSN 0133-3720 R&D Projects: GA ČR(CZ) GPP503/12/P395 Institutional support: RVO:68081715 Keywords : barley * wheat * glycoprotein * mass spectrometry * lectin chromatography Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 0.496, year: 2016

  8. Humanizing recombinant glycoproteins from Chinese hamster ovary cells

    DEFF Research Database (Denmark)

    Hansen, Anders Holmgaard; Amann, Thomas; Kol, Stefan

    hamster ovary (CHO) cells are making a very heterogeneous mixture of NGlycans. We speculate that the CHO pattern of N-Glycans would affect half-life and/or efficacy of the glycoprotein in the bloodstream making it unsuitable for human intravenous use, whereas our humanized version would be identical...

  9. Molecular cloning of S1 glycoprotein gene of infectious bronchitis ...

    African Journals Online (AJOL)

    In vitro protein expression is an important method of obtaining large amounts of viral proteins to investigate their biological properties. The S1 glycoprotein of infectious bronchitis virus, due to its effective immune-dominant role is an appropriate candidate for production of recombinant vaccine against infectious bronchitis ...

  10. Separation and identification of carp pituitary proteins and glycoproteins

    Czech Academy of Sciences Publication Activity Database

    Ryšlavá, H.; Janatová, M.; Čalounová, G.; Selicharová, Irena; Barthová, J.; Barth, Tomislav

    2005-01-01

    Roč. 50, č. 9 (2005), 430-437 ISSN 1212-1819 R&D Projects: GA MZe(CZ) QF3028 Institutional research plan: CEZ:AV0Z4055905 Keywords : carp hormones * glycoproteins * oligosaccharide chains Subject RIV: CE - Biochemistry Impact factor: 0.254, year: 2005

  11. QUANTITATIVE MASS SPECTROMETRIC ANALYSIS OF GLYCOPROTEINS COMBINED WITH ENRICHMENT METHODS

    Science.gov (United States)

    Ahn, Yeong Hee; Kim, Jin Young; Yoo, Jong Shin

    2015-01-01

    Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Rapid Commun. Mass Spec Rev 34:148–165, 2015. PMID:24889823

  12. Increasing nerve agent treatment efficacy by P-glycoprotein inhibition

    NARCIS (Netherlands)

    Joosen, M.J.A.; Vester, S.M.; Hamelink, J.; Klaassen, S.D.; Berg, R.M. van den

    2016-01-01

    One of the shortcomings of current treatment of nerve agent poisoning is that not all drugs effectively penetrate the blood-brain barrier (BBB), whereas most nerve agents easily do. P-glycoprotein (Pgp) efflux transporters at the BBB may contribute to this aspect. It was previously shown that Pgp

  13. Glycoprotein Ibalpha signalling in platelet apoptosis and clearance

    NARCIS (Netherlands)

    van der Wal, E.

    2010-01-01

    Storage of platelets at low temperature reduces bacterial growth and might better preserve the haemostatic function of platelets than current procedures. Incubation at 0C is known to expose ?-N-acetyl-D-glucosamine-residues on glycoprotein (GP)Ibalpha inducing receptor-clustering and platelet

  14. Glycoprotein Ibα clustering in platelet storage and function

    NARCIS (Netherlands)

    Gitz, E.

    2013-01-01

    Platelets are anucleated, discoid-shaped cells that play an essential role in the formation of a hemostatic plug to prevent blood loss from injured vessels. Initial platelet arrest at the damaged arterial vessel wall is mediated through the interaction between the platelet receptor glycoprotein (GP)

  15. Mechanism for maturation-related reorganization of flavivirus glycoproteins.

    Science.gov (United States)

    Plevka, Pavel; Battisti, Anthony J; Sheng, Ju; Rossmann, Michael G

    2014-01-01

    Flaviviruses, such as dengue, West Nile, and yellow fever viruses, assemble as fusion-incompetent particles and subsequently undergo a large reorganization of their glycoprotein envelope resulting in formation of mature infectious virions. Here we used a combination of three-dimensional cryo-electron tomography and two-dimensional image analysis to study pleomorphic maturation intermediates of dengue virus 2. Icosahedral symmetries of immature and mature regions within one particle were mismatched relative to each other. Furthermore, the orientation of the two regions relative to each other differed among particles. Therefore, there cannot be a specific pathway determining the maturation of all particles. Instead, the region with mature structure expands when glycoproteins on its boundary acquire suitable orientation and conformation to allow them to become a stable part of the mature region. This type of maturation is possible because the envelope glycoproteins are anchored to the phospholipid bilayer that is a part of flavivirus virions and are thus restricted to movement on the two-dimensional surface of the particle. Therefore, compounds that limit movement of the glycoproteins within the virus membrane might be used as inhibitors of flavivirus maturation. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Do N-glycoproteins have preference for specific sequons?

    DEFF Research Database (Denmark)

    Rao, Shyama Prasad; Wollenweber, Bernd

    2010-01-01

    (hemagglutinin of influenza A H3N2 and glycoprotein120 of HIV-1) are indeed preferred sequon types, which may provide a selective advantage. Accordingly, although there seems to be some preference for sequons, this preference may not be unique to N-glycosylation....

  17. Extra-oviductal expression of oviductal glycoprotein 1 in mouse ...

    Indian Academy of Sciences (India)

    J. Biosci. 42(1), March 2017, 69–80 * Indian Academy of Sciences. 69. DOI: 10.1007/s12038-016-9657-2. Keywords. Epididymis; ovary; oviductal glycoprotein 1; testis. Supplementary materials pertaining to this article are available on the Journal of Biosciences Website. Published online: 11 January 2017 ...

  18. Platelet glycoproteins and fibrinogen in recovery from idiopathic sudden hearing loss.

    Directory of Open Access Journals (Sweden)

    Daniel Weiss

    Full Text Available BACKGROUND: The pathomechanism and location of idiopathic sudden sensorineural hearing loss (ISSHL is unclear. In a previous case-control study, we found elevated fibrinogen concentrations and a higher prevalence of T allele carriers of the glycoprotein (Gp Ia C807T polymorphism in ISSHL patients. METHODOLOGY: 127 patients with ISSHL (mean age 53.3 years, 48.8% females, who underwent a standard therapy with high dose steroids, pentoxifyllin and sterofundine over 8 days were included. We examined the influence of GpIa genotype and fibrinogen (BclI-, A312-, HaeIII- genotype and fibrinogen plasma levels on hearing recovery after 8 weeks (change from baseline: 0 dB  =  no recovery, >0 to 10 dB = moderate recovery, >10 dB = good recovery. In a subsample of 59 patients with ISSHL, we further studied the association of platelet glycoprotein GpIa, Ib and IIIa densities on hearing recovery as well as the possible effect-modification of platelet glycoproteins on hearing recovery by plasma fibrinogen. RESULTS: In univariate analysis, neither the GpIa genotype nor fibrinogen genotype (all p>0.1 but lower fibrinogen levels (p = 0.029, less vertigo (p = 0.002 and lower GpIIIa receptor density (p = 0.037, n = 59 were associated with hearing recovery. In multivariate analysis, fibrinogen significantly modified the effect of GPIa receptor density on good hearing recovery (effect-modification on multiplicative scale OR = 0.45 (95% confidence interval (0.21-0.94, p = 0.03. GPIb receptor density below the mean was associated with a 2-fold increase in good hearing recovery both in patients with fibrinogen levels above (p = 0.04 as well as in patients with fibrinogen levels below the mean (p = 0.06. There was no indication for an effect-modification (p = 0.97. CONCLUSIONS: The findings suggest a vascular/rheological origin of ISSHL with unique features of thrombosis in the inner ear artery that may include complex

  19. Shedding of soluble glycoprotein 1 detected during acute Lassa virus infection in human subjects

    Directory of Open Access Journals (Sweden)

    Momoh Mambu

    2010-11-01

    Full Text Available Abstract Background Lassa hemorrhagic fever (LHF is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. With a high rate of infection that may lead to morbidity and mortality, understanding how the virus interacts with the host's immune system is of great importance for generating vaccines and therapeutics. Previous work by our group identified a soluble isoform of the Lassa virus (LASV GP1 (sGP1 in vitro resulting from the expression of the glycoprotein complex (GPC gene 12. Though no work has directly been done to demonstrate the function of this soluble isoform in arenaviral infections, evidence points to immunomodulatory effects against the host's immune system mediated by a secreted glycoprotein component in filoviruses, another class of hemorrhagic fever-causing viruses. A significant fraction of shed glycoprotein isoforms during viral infection and biogenesis may attenuate the host's inflammatory response, thereby enhancing viral replication and tissue damage. Such shed glycoprotein mediated effects were previously reported for Ebola virus (EBOV, a filovirus that also causes hemorrhagic fever with nearly 90% fatality rates 345. The identification of an analogous phenomenon in vivo could establish a new correlate of LHF infection leading to the development of sensitive diagnostics targeting the earliest molecular events of the disease. Additionally, the reversal of potentially untoward immunomodulatory functions mediated by sGP1 could potentiate the development of novel therapeutic intervention. To this end, we investigated the presence of sGP1 in the serum of suspected LASV patients admitted to the Kenema Government Hospital (KGH Lassa Fever Ward (LFW, in Kenema, Sierra Leone that tested positive for viral antigen or displayed classical signs of Lassa fever. Results It is reasonable to expect that a narrow window exists for

  20. Characterisation of the carbohydrate components of Taenia solium metacestode glycoprotein antigens.

    Science.gov (United States)

    Restrepo, B I; Obregón-Henao, A; Mesa, M; Gil, D L; Ortiz, B L; Mejía, J S; Villota, G E; Sanzón, F; Teale, J M

    2000-05-01

    Human neurocysticercosis is caused by Taenia solium metacestodes. It usually affects the central nervous system of humans and can be confused with other brain pathologies. The Lens culinaris-binding glycoproteins from this parasite have been shown to be ideal targets for the development of a highly specific immunoassay for the diagnosis of neurocysticercosis. In the present study we characterised the carbohydrates associated with five antigenic glycoproteins of T. solium metacestodes in the range of 12-28 kilodaltons. Lectin-affinities and enzymatic deglycosylations suggested that each of the five antigens contain various glycoforms of asparagine-linked carbohydrates of the hybrid, complex and probably high mannose type. These carbohydrates accounted for at least 30-66% of the apparent molecular mass of the glycoconjugates. In contrast, there was no evidence for the presence of O-linked carbohydrates. Lectin affinity patterns suggested that the sugars are short and truncated in their biosynthetic route, and that some contain terminal galactose moieties. Elucidating the precise structure of the carbohydrates and establishing their role in antigenicity will be essential to design strategies to produce them in large and reproducible amounts for the development of improved immunoassays.

  1. The HIV-1 envelope glycoprotein structure: nailing down a moving target.

    Science.gov (United States)

    Ward, Andrew B; Wilson, Ian A

    2017-01-01

    Structure determination of the HIV-1 envelope glycoprotein (Env) presented a number of challenges, but several high-resolution structures have now become available. In 2013, cryo-EM and x-ray structures of soluble, cleaved SOSIP Env trimers from the clade A BG505 strain provided the first glimpses into the Env trimer fold as well as more the variable regions. A recent cryo-EM structure of a native full-length trimer without any stabilizing mutations had the same core structure, but revealed new insights and features. A more comprehensive and higher resolution understanding of the glycan shield has also emerged, enabling a more complete representation of the Env glycoprotein structure. Complexes of Env trimers with broadly neutralizing antibodies have surprisingly illustrated that most of the Env surface can be targeted in natural infection and that the neutralizing epitopes are almost all composed of both peptide and glycan components. These structures have also provided further evidence of the inherent plasticity of Env and how antibodies can exploit this flexibility by perturbing or even stabilizing the trimer to facilitate neutralization. These breakthroughs have stimulated further design and stabilization of Env trimers as well as other platforms to generate trimers that now span multiple subtypes. These Env trimers when used as immunogens, have led to the first vaccine-induced neutralizing antibodies for structural and functional analyses. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity.

    Science.gov (United States)

    Furihata, K; Clemetson, K J; Deguchi, H; Kunicki, T J

    2001-11-01

    - Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

  3. Platelet receptor expression and shedding: glycoprotein Ib-IX-V and glycoprotein VI.

    Science.gov (United States)

    Gardiner, Elizabeth E; Andrews, Robert K

    2014-04-01

    Quantity, quality, and lifespan are 3 important factors in the physiology, pathology, and transfusion of human blood platelets. The aim of this review is to discuss the proteolytic regulation of key platelet-specific receptors, glycoprotein(GP)Ib and GPVI, involved in the function of platelets in hemostasis and thrombosis, and nonimmune or immune thrombocytopenia. The scope of the review encompasses the basic science of platelet receptor shedding, practical aspects related to laboratory analysis of platelet receptor expression/shedding, and clinical implications of using the proteolytic fragments as platelet-specific biomarkers in vivo in terms of platelet function and clearance. These topics can be relevant to platelet transfusion regarding both changes in platelet receptor expression occurring ex vivo during platelet storage and/or clinical use of platelets for transfusion. In this regard, quantitative analysis of platelet receptor profiles on blood samples from individuals could ultimately enable stratification of bleeding risk, discrimination between causes of thrombocytopenia due to impaired production vs enhanced clearance, and monitoring of response to treatment prior to change in platelet count. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. A variant surface glycoprotein of Trypanosoma brucei is synthesized with a hydrophobic carboxy-terminal extension from purified glycoprotein.

    NARCIS (Netherlands)

    J.C. Boothroyd; G.A.M. Cross; J.H.J. Hoeijmakers (Jan); P. Borst (Piet)

    1980-01-01

    textabstractSequential expression of variant surface glycoproteins (VSGs) enables the parasitic protozoan Trypanosoma brucei to evade the immune response of its mammalian hosts. Studies of several VSGs, which have been isolated as soluble molecules following disruption of cells in the absence of

  5. Cloning of partial cDNA encoding differentiation and tumor-associated mucin glycoproteins expressed by human mammary epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Gender, S.J.; Burchell, J.M.; Duhig, T.; Lamport, D.; White, R.; Parker, M.; Taylor-Papadimitriou, J.

    1987-09-01

    Human mammary epithelial cells secrete and express on their cell surfaces complex mucin glycoproteins that are developmentally regulated, tumor-associated, and highly immunogenic. Studies using monoclonal antibodies directed to these glycoproteins suggest that their molecular structures can vary with differentiation stages in the normal gland and in malignancy. To analyze the molecular nature of these glycoproteins, milk mucin was affinity-purifed and deglycosylated with hydrogen fluoride, yielding bands at 68 and 72 kDa on silver-stained gels. Polyclonal and monoclonal antibodies to the stripped core protein were developed and used to screen a lambdagt11 expression library of cDNA made from mRNA of the mammary tumor cell line MCF-7. Seven crossreacting clones were isolated, with inserts 0.1-1.8 kilobases long. RNA blot analysis, using as a probe the 1.8-kilobase insert subcloned in plasmid pUC8 (pMUC10), revealed transcripts of 4.7 and 6.4 kilobases in MCF-7 and T47D mammary tumor cells, whereas normal mammary epithelial cells from pooled milks have additional transcripts. The expression of mRNA correlates with antigen expression as determined by binding of two previously characterized anti-mucin monoclonal antibodies (HMFG-1 and HMFG-2) to seven cell lines. Restriction enzyme analysis detected a restriction fragment length polymorphism when human genomic DNA was digested with EcoRI or HinfI.

  6. Ice growth in supercooled solutions of antifreeze glycoprotein.

    Science.gov (United States)

    Harrison, K; Hallett, J; Burcham, T S; Feeney, R E; Kerr, W L; Yeh, Y

    Inhibition of ice growth in supercooled solution by certain proteins is vital to the survival of many living organisms. Some fish, native to both subzero northern and southern waters, have special proteins or glycoproteins in their blood serum that inhibit ice formation. Whereas these proteins have only a very small effect on the melting temperature of ice, the temperature of these fish can fall to nearly 1 K below the melting point before ice crystals grow. This phenomenon is called freezing hysteresis, in contrast to the normal colligative effect of solutes that depresses the equilibrium temperature, around which small changes lead to crystal growth or melting depending on sign. Some insects also exhibit a serum freezing hysteresis. We report the effects of different degrees of supercooling on the habit and rates of growth of ice crystals from solutions of these antifreeze glycoproteins (AFGPs). We find that the crystallization rate is up to five times greater than that in pure water.

  7. TROPHOBLASTIC β1 – GLYCOPROTEIN SYNTHESIS IN SEROPOSITIVE PREGNANT WOMEN

    Directory of Open Access Journals (Sweden)

    R. N. Bogdanovich

    2005-01-01

    Full Text Available Abstract. The level of trophoblastic β1 – glycoprotein (SP–1 was determined in the blood sera of 200 healthy pregnant women and 184 women with threatened abortions in term till 20 weeks of pregnancy. In group of women experiencing recurrent abortions in 38 % cases antibodies to chorionic gonadotropin, in 39,5 % cases antibodies to phospholipids, in 25,5 % – antibodies to tireoglobulin were revealed in significant amounts. In 20,65 % lupus anticoagulant was found. The majority of women in this group had changes in homeostasis. The presence of autoantibodies during pregnancy is the unfavourable factor in the development of placental insufficiency. This is proved by the decreased secretion of trophoblastic β1 – glycoprotein – a marker of the fetal part of placenta. (Med. Immunol., 2005, vol.7, № 1, pp. 85588

  8. Comparison of glycoprotein expression between ovarian and colon adenocarcinomas

    DEFF Research Database (Denmark)

    Multhaupt, H A; Arenas-Elliott, C P; Warhol, M J

    1999-01-01

    , carcinoembryonic antigen, and cytokeratins 7 and 20 to detect tumor-associated glycoproteins and keratin proteins in ovarian and colonic carcinomas. RESULTS: CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 can distinguish between colonic and serous or endometrioid adenocarcinomas of the ovary in both...... primary and metastatic lesions. Mucinous ovarian adenocarcinomas differed in that they express carcinoembryonic antigen and cytokeratins 7 and 20 and weakly express CA125. The other glycoprotein antigens were equally expressed by ovarian and colonic adenocarcinomas and therefore were of no use...... in distinguishing between these 2 entities. CONCLUSION: A panel of monoclonal antibodies against cytokeratins 7 and 20 antigens, CA125, and carcinoembryonic antigen is useful in differentiating serous and endometrioid adenocarcinomas of the ovary from colonic adenocarcinomas. Mucinous ovarian adenocarcinomas cannot...

  9. Variable Lymphocyte Receptor Recognition of the Immunodominant Glycoprotein of Bacillus anthracis Spores

    Energy Technology Data Exchange (ETDEWEB)

    Kirchdoerfer, Robert N.; Herrin, Brantley R.; Han, Byung Woo; Turnbough, Jr., Charles L.; Cooper, Max D.; Wilson, Ian A. (SNU); (Scripps); (Emory); (UAB); (Emory Vaccine)

    2012-07-25

    Variable lymphocyte receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin fold-based T and B cell receptors, lymphocyte-like cells of jawless fish express VLRs (VLRA, VLRB, or VLRC) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA and VLRC) in function. Here, we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis, and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores.

  10. The macrophage CD163 surface glycoprotein is an erythroblast adhesion receptor

    DEFF Research Database (Denmark)

    Fabriek, Babs O; Polfliet, Machteld M J; Vloet, Rianka P M

    2007-01-01

    on the surface of macrophages in erythroblastic islands, in erythroblast binding. In particular, the monoclonal antibody ED2 was found to inhibit erythroblast binding to bone marrow macrophages. Here, we identify the ED2 antigen as the rat CD163 surface glycoprotein, a member of the group B scavenger receptor...... cysteine-rich (SRCR) family that has previously been shown to function as a receptor for hemoglobin-haptoglobin (Hb-Hp) complexes and is believed to contribute to the clearance of free hemoglobin. CD163 transfectants and recombinant protein containing the extracellular domain of CD163 supported...... the adhesion of erythroblastic cells. Furthermore, we identified a 13-amino acid motif (CD163p2) corresponding to a putative interaction site within the second scavenger receptor domain of CD163 that could mediate erythroblast binding. Finally, CD163p2 promoted erythroid expansion in vitro, suggesting...

  11. Alpha1-acid glycoprotein post-translational modifications: a comparative two dimensional electrophoresis based analysis

    Directory of Open Access Journals (Sweden)

    P. Roncada

    2010-04-01

    Full Text Available Alpha1-acid glycoprotein (AGP is an immunomodulatory protein expressed by hepatocytes in response to the systemic reaction that follows tissue damage caused by inflammation, infection or trauma. A proteomic approach based on two dimensional electrophoresis, immunoblotting and staining of 2DE gels with dyes specific for post-translational modifications (PTMs such as glycosylation and phosphorylation has been used to evaluate the differential interspecific protein expression of AGP purified from human, bovine and ovine sera. By means of these techniques, several isoforms have been identified in the investigated species: they have been found to change both with regard to the number of isoforms expressed under physiological condition and with regard to the quality of PTMs (i.e. different oligosaccharidic chains, presence/absence of phosphorilations. In particular, it is suggested that bovine serum AGP may have one of the most complex pattern of PTMs among serum proteins of mammals studied so far.

  12. Tumor specific glycoproteins and method for detecting tumorigenic cancers

    International Nuclear Information System (INIS)

    Davidson, E.A.; Bolmer, S.D.

    1981-01-01

    The detection of tumour specific glycoproteins (TSGP) in human sera often indicates the presence of a malignant tumour in a patient. The distinguishing characteristics of TSGP isolated from the blood sera of cancer patients are described in detail together with methods of TSGP isolation and purification. Details are also given of radioimmunoassay techniques capable of detecting very low levels of serum TSGP with high specificity. (U.K.)

  13. Mucus glycoprotein secretion by tracheal explants: effects of pollutants

    International Nuclear Information System (INIS)

    Last, J.A.; Kaizu, T.

    1980-01-01

    Tracheal slices incubated with radioactive precursors in tissue culture medium secrete labeled mucus glycoproteins into the culture medium. We have used an in vivtro approach, a combined method utilizing exposure to pneumotoxins in vivo coupled with quantitation of mucus secretion rates in vitro, to study the effects of inhaled pollutants on mucus biosynthesis by rat airways. In addition, we have purified the mucus glycoproteins secreted by rat tracheal explants in order to determine putative structural changes that might by the basis for the observed augmented secretion rates after exposure of rats to H2SO4 aerosols in combination with high ambient levels of ozone. After digestion with papain, mucus glycoproteins secreted by tracheal explants may be separated into five fractions by ion-exchange chromatography, with recovery in high yield, on columns of DEAE-cellulose. Each of these five fractions, one neutral and four acidic, migrates as a single unique spot upon cellulose acetate electrophoresis at pH values of 8.6 and 1.2. The neutral fraction, which is labeled with [3H] glucosamine, does not contain radioactivity when Na2 35SO4 is used as the precursor. Acidic fractions I to IV are all labeled with either 3H-glucosamine or Na2 35SO4 as precursor. Acidic fraction II contains sialic acid as the terminal sugar on its oligosaccharide side chains, based upon its chromatographic behavior on columns of wheat-germ agglutinin-Agarose. Treatment of this fraction with neuraminidase shifts its elution position in the gradient to a lower salt concentration, coincident with acidic fraction I. After removal of terminal sialic acid residues with either neuraminidase or low pH treatment, the resultant terminal sugar on the oligosaccharide side chains is fucose. These results are identical with those observed with mucus glycoproteins secreted by cultured human tracheal explants and purified by these same techniques

  14. Prediction of conserved sites and domains in glycoproteins B, C and D of herpes viruses.

    Science.gov (United States)

    Rasheed, Muhammad Asif; Ansari, Abdur Rahman; Ihsan, Awais; Navid, Muhammad Tariq; Ur-Rehman, Shahid; Raza, Sohail

    2018-03-01

    Glycoprotein B (gB), C (gC) and D (gD) of herpes simplex virus are implicated in virus adsorption and penetration. The gB, gC and gD are glycoproteins for different processes of virus binding and attachment to the host cells. Moreover, their expression is necessary and sufficient to induce cell fusion in the absence of other glycoproteins. Egress of herpes simplex virus (HSV) and other herpes viruses from cells involves extensive modification of cellular membranes and sequential envelopment, de-envelopment and re-envelopment steps. Viral glycoproteins are important in these processes, and frequently two or more glycoproteins can largely suffice in any step. Hence, we target the 3 important glycoproteins (B, C and D) of eight different herpes viruses of different species. These species include human (HSV1 and 2), bovine (BHV1), equine (EHV1 and 4), chicken (ILT1 and MDV2) and pig (PRV1). By applying different bioinformatics tools, we highlighted the conserved sites in these glycoproteins which might be most significant regarding attachment and infection of the viruses. Moreover the conserved domains in these glycoproteins are also highlighted. From this study, we will able to analyze the role of different viral glycoproteins of different species during herpes virus adsorption and penetration. Moreover, this study will help to construct the antivirals that target the glycoproteins of different herpes viruses. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Glycoprotein fucosylation is increased in seminal plasma of subfertile men

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    Beata Olejnik

    2015-04-01

    Full Text Available Fucose, the monosaccharide frequent in N- and O-glycans, is a part of Lewis-type antigens that are known to mediate direct sperm binding to the zona pellucida. Such interaction was found to be inhibited in vitroby fucose-containing oligo- and polysaccharides, as well as neoglycoproteins. The objective of this study was to screen seminal plasma proteins of infertile/subfertile men for the content and density of fucosylated glycoepitopes, and compare them to samples of fertile normozoospermic subjects. Seminal proteins were separated in polyacrylamide gel electrophoresis and blotted onto nitrocellulose membrane and probed with fucose-specific Aleuria aurantia lectin (AAL. Twelve electrophoretic bands were selected for quantitative densitometric analysis. It was found that the content, and especially the density of fucosylated glycans, were higher in glycoproteins present in seminal plasma of subfertile men. No profound differences in fucosylation density were found among the groups of normozoospermic, oligozoospermic, asthenozoospermic, and oligoasthenozoospermic subfertile men. According to the antibody probing, AAL-reactive bands can be attributed to male reproductive tract glycoproteins, including prostate-specific antigen, prostatic acid phosphatase, glycodelin and chorionic gonadotropin. Fibronectin, α1 -acid glycoprotein, α1 -antitrypsin, immunoglobulin G and antithrombin III may also contribute to this high fucosylation. It is suggested that the abundant fucosylated glycans in the sperm environment could interfere with the sperm surface and disturb the normal course of the fertilization cascade.

  16. Extracellular Glycoproteins in Embryogenic Culture of Pumpkin (Cucurbita pepo L.

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    Hana Čipčić Paljetak

    2011-01-01

    Full Text Available The extracellular proteins in three distinctly induced embryogenic lines of pumpkin (Cucurbita pepo L. cultivated in four MS media modified regarding the nitrogen composition or auxin presence/absence have been analyzed. Extracellular glycoproteins containing α-D-mannose were specifically detected by the lectine concavalin A. During the cultivation of embryogenic tissue in the medium supplemented with reduced nitrogen, the embryos were mostly arrested at preglobular and globular developmental stages, which coincide with the absence of protein secretion. Secreted glycoproteins of 76, 68, 37 and 34 kDa were detected only if any of the three lines were cultivated in the medium that stimulates embryo development, irrespectively of the addition of 2,4-dichlorophenoxyacetic acid or tunicamycin. The glycoprotein of 64 kDa was detected in all lines cultivated in hormone-free MS medium with conventional nitrogen sources and it appears to be associated with embryo maturation. Tunicamycin treatment did not influence embryogenesis, although it specifically affected glycosylation of proteins in the investigated lines. Our results show that besides auxin, the source of nitrate is of great importance for proper protein glycosylation, excretion and developmental transition of pumpkin somatic embryos.

  17. Comparison of glycoprotein expression between ovarian and colon adenocarcinomas.

    Science.gov (United States)

    Multhaupt, H A; Arenas-Elliott, C P; Warhol, M J

    1999-10-01

    Tumor-associated antigens may be expressed as surface glycoproteins. These molecules undergo qualitative and quantitative modifications during cell differentiation and malignant transformation. During malignant transformation, incomplete glycosylation is common, and certain glycosylation pathways are preferred. These antigens might help distinguish between ovarian and colonic adenocarcinomas in the primary and metastatic lesions. Different cytokeratins have been proposed as relatively organ-specific antigens. We used monoclonal antibodies against T1, Tn, sialosyl-Tn, B72.3, CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 to detect tumor-associated glycoproteins and keratin proteins in ovarian and colonic carcinomas. CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 can distinguish between colonic and serous or endometrioid adenocarcinomas of the ovary in both primary and metastatic lesions. Mucinous ovarian adenocarcinomas differed in that they express carcinoembryonic antigen and cytokeratins 7 and 20 and weakly express CA125. The other glycoprotein antigens were equally expressed by ovarian and colonic adenocarcinomas and therefore were of no use in distinguishing between these 2 entities. A panel of monoclonal antibodies against cytokeratins 7 and 20 antigens, CA125, and carcinoembryonic antigen is useful in differentiating serous and endometrioid adenocarcinomas of the ovary from colonic adenocarcinomas. Mucinous ovarian adenocarcinomas cannot be distinguished from colonic adenocarcinomas using immunohistochemistry.

  18. Australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing

    Energy Technology Data Exchange (ETDEWEB)

    Tropea, J.E.; Molyneux, R.J.; Kaushal, G.P.; Pan, Y.T.; Mitchell, M.; Elbein, A.D. (Univ. of Texas Health Science Center, San Antonio (USA))

    1989-03-07

    Australine is a polyhydroxylated pyrrolizidine alkaloid that was isolated from the seeds of the Australian tree Castanospermum australe and characterized by NMR and X-ray diffraction analysis. Since swainsonine and catanospermine are polyhydroxylated indolizidine alkaloids that inhibit specific glycosidases, the authors tested australine against a variety of exoglycosidases to determine whether it would inhibit any of these enzymes. This alkaloid proved to be a good inhibitor of the {alpha}-glucosidase amyloglucosidase (50% inhibition at 5.8 {mu}M), but it did not inhibit {beta}-glucosidase, {alpha}- or {beta}-mannosidase, or {alpha}- or {beta}-galactosidase. The inhibition of amyloglucosidase was of a competitive nature. Australine also inhibited the glycoprotein processing enzyme glucosidase I, but had only slight activity toward glucosidase II. When incubated with cultured cells, this alkaloid inhibited glycoprotein processing at the glucosidase I step and caused the accumulation of glycoproteins with Glc{sub 3}Man{sub 7-9}(GlcNAc){sub 2}-oligosaccharides.

  19. Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates.

    Science.gov (United States)

    Kwong, P D; Wyatt, R; Majeed, S; Robinson, J; Sweet, R W; Sodroski, J; Hendrickson, W A

    2000-12-15

    The gp120 exterior envelope glycoprotein of HIV-1 binds sequentially to CD4 and chemokine receptors on cells to initiate virus entry. During natural infection, gp120 is a primary target of the humoral immune response, and it has evolved to resist antibody-mediated neutralization. We previously reported the structure at 2.5 A of a gp120 core from the HXBc2 laboratory-adapted isolate in complex with a 2 domain fragment of CD4 and the antigen binding fragment of a human antibody. This revealed atomic details of gp120-receptor interactions and suggested multiple mechanisms of immune evasion. We have now extended the HXBc2 structure in P222, crystals to 2.2 A. The enhanced resolution enabled a more accurate modeling of less-well-ordered regions and provided conclusive identification of the density in the central cavity at the crux of the gp120-CD4 interaction as isopropanol from the crystallization medium. We have also determined the structure of a gp120 core from the primary clinical HIV-1 isolate, YU2, in the same ternary complex but in a C2 crystal lattice. Comparisons of HXBc2 and YU2 showed that while CD4 binding was rigid, portions of the gp120 core were conformationally flexible; overall differences were minor, with sequence changes concentrated on a surface expected to be exposed on the envelope oligomer. Despite dramatic antigenic differences between primary and laboratory-adapted HIV-1, the gp120 cores from these isolates are remarkably similar. Taken together with chimeric substitution and sequence analysis, this indicates that neutralization resistance is specified by quaternary interactions involving the major variable loops and thus affords a mechanism for viral adaptation. Conservation of the central cavity suggests the possibility of therapeutic inhibitors. The structures reported here extend in detail and generality our understanding of the biology of the gp120 envelope glycoprotein.

  20. Production of glycoprotein vaccines in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ihssen Julian

    2010-08-01

    Full Text Available Abstract Background Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries. Results Two different periplasmic carrier proteins, AcrA from C. jejuni and a toxoid form of Pseudomonas aeruginosa exotoxin were glycosylated with Shigella O antigens in E. coli. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in E. coli. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of E. coli cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold. Conclusions The presented data demonstrate that glycosylated proteins can be produced in recombinant E. coli at a larger scale. The described methodologies constitute an important step

  1. Spatial distribution and molecular dynamics of dystrophin glycoprotein components at the neuromuscular junctionin vivo.

    Science.gov (United States)

    Aittaleb, Mohamed; Martinez-Pena Y Valenzuela, Isabel; Akaaboune, Mohammed

    2017-05-15

    A bimolecular fluorescence complementation (BiFC) approach was used to study the molecular interactions between different components of the postsynaptic protein complex at the neuromuscular junction of living mice. We show that rapsyn forms complex with both α-dystrobrevin and α-syntrophin at the crests of junctional folds. The linkage of rapsyn to α-syntrophin and/or α-dystrobrevin is mediated by utrophin, a protein localized at acetylcholine receptor (AChR)-rich domains. In mice deficient in α-syntrophin, in which utrophin is no longer present at the synapse, rapsyn interaction with α-dystrobrevin was completely abolished. This interaction was completely restored when either utrophin or α-syntrophin was introduced into muscles deficient in α-syntrophin. However, in neuromuscular junctions deficient in α-dystrobrevin, in which utrophin is retained, complex formation between rapsyn and α-syntrophin was unaffected. Using fluorescence recovery after photobleaching, we found that α-syntrophin turnover is 5-7 times faster than that of AChRs, and loss of α-dystrobrevin has no effect on rapsyn and α-syntrophin half-life, whereas the half-life of AChR was significantly altered. Altogether, these results provide new insights into the spatial distribution of dystrophin glycoprotein components and their dynamics in living mice. © 2017. Published by The Company of Biologists Ltd.

  2. Monoclonal antibodies directed to E1 glycoprotein of rubella virus

    International Nuclear Information System (INIS)

    Umino, Y.; Sato, A.; Katow, S.; Matsuno, T.; Sugiura, A.

    1985-01-01

    We have prepared four monoclonal antibodies to rubella virus E1 glycoprotein. Three nonoverlapping antigenic sites were delineated on E1 protein by competitive binding assays. Antibodies binding to one site were characterized by high hemagglutination inhibition (HI) titer but poor neutralizing activity. The addition of antiglobulin conferred neutralizing activity. Antibodies directed to two other antigenic sites had modest hemolysis inhibition but little or no HI and neutralizing activities. The addition of antiglobulin markedly augmented HI activity but had little effect on neutralizing activity. Epitopes defined by three antibodies were conserved among four rubella virus strains examined. (Author)

  3. The COMPLEXity in herpesvirus entry.

    Science.gov (United States)

    Sathiyamoorthy, Karthik; Chen, Jia; Longnecker, Richard; Jardetzky, Theodore S

    2017-06-01

    Enveloped viruses have evolved diverse transmembrane proteins and protein complexes to enable host cell entry by regulating and activating membrane fusion in a target cell-specific manner. In general terms, the entry process requires a receptor binding step, an activation step and a membrane fusion step, which can be encoded within a single viral protein or distributed among multiple viral proteins. HIV and influenza virus, for example, encode all of these functions in a single trimeric glycoprotein, HIV env or influenza virus hemagglutinin (HA). In contrast, herpesviruses have the host receptor binding, activation and fusogenic roles distributed among multiple envelope glycoproteins (ranging from three to six), which must coordinate their functions at the site of fusion. Despite the apparent complexity in the number of viral entry proteins, herpesvirus entry is fundamentally built around two core glycoprotein entities: the gHgL complex, which appears to act as an 'activator' of entry, and the gB protein, which is thought to act as the membrane 'fusogen'. Both are required for all herpesvirus fusion and entry. In many herpesviruses, gHgL either binds host receptors directly or assembles into larger complexes with additional viral proteins that bind host receptors, conferring specificity to the cells that are targeted for infection. These gHgL entry complexes (ECs) are centrally important to activating gB-mediated membrane fusion and establishing viral tropism, forming membrane bridging intermediates before gB triggering. Here we review recent structural and functional studies of Epstein-Barr virus (EBV) and Cytomegalovirus (CMV) gHgL complexes that provide a framework for understanding the role of gHgL in herpesvirus entry. Furthermore, a recently determined EM model of Herpes Simplex virus (HSV) gB embedded in exosomes highlights how gB conformational changes may promote viral and cellular membrane fusion. Copyright © 2017. Published by Elsevier B.V.

  4. Thrombasthenie de Glanzmann et grossesse: a propos d�un cas et revue de la litterature

    Directory of Open Access Journals (Sweden)

    Myriam Rachad

    2011-12-01

    Full Text Available La thrombasthenie de Glanzmann est une anomalie plaquettaire qualitative, a transmission autosomale recessive, due a un deficit en glycoproteine IIb/IIIa (GPIIb/IIIa, avec un defaut d�agregation plaquettaire. Il en resulte une perturbation de l�hemostase primaire, a l�origine d�hemorragie plus ou mois importantes. Nous rapportons l�observation de N.A, agee de 32 ans, suivie depuis l�enfance pour maladie de Glanzmann, troisieme pare, avec deux accouchements par voie basse et une cesarienne programmee, compliques tous les trois par hemorragie de delivrance, jugulee avec succes. La grossesse chez une patiente thrombo-asthenique reste rare, peu de cas jusqu'a present, sont decrits. A travers cette observation, et a la lumiere d�une revue de la litterature, nous allons essayer surtout, de mettre le point sur la prise en charge de cette association delicate, qui met en jeu, a chaque fois, le pronostic vital maternel.

  5. Effects of cobalt-chromium everolimus eluting stents or bare metal stent on fatal and non-fatal cardiovascular events

    DEFF Research Database (Denmark)

    Valgimigli, Marco; Sabaté, Manel; Kaiser, Christoph

    2014-01-01

    eluting stents with bare metal stents were selected. The principal investigators whose trials met the inclusion criteria provided data for individual patients. PRIMARY OUTCOMES: The primary outcome was cardiac mortality. Secondary endpoints were myocardial infarction, definite stent thrombosis, definite...... a significant reduction of cardiac mortality (hazard ratio 0.67, 95% confidence interval 0.49 to 0.91; P=0.01), myocardial infarction (0.71, 0.55 to 0.92; P=0.01), definite stent thrombosis (0.41, 0.22 to 0.76; P=0.005), definite or probable stent thrombosis (0.48, 0.31 to 0.73; P... coronary syndrome v stable coronary artery disease), diabetes mellitus, female sex, use of glycoprotein IIb/IIIa inhibitors, and up to one year v longer duration treatment with dual antiplatelets. CONCLUSIONS: This meta-analysis offers evidence that compared with bare metal stents the use of cobalt...

  6. Reduced platelet activation and platelet aggregation in patients with alcoholic liver cirrhosis

    DEFF Research Database (Denmark)

    Vinholt, Pernille Just; Hvas, Anne-Mette; Nielsen, Christian

    2018-01-01

    in stimulated samples were lower in patients versus healthy individuals, e.g., after collagen-related peptide stimulation, the median percentage of platelets positive for activated glycoprotein IIb/IIIa was 85% (IQR 70-94) in patients versus 97% (IQR 94-99) in healthy individuals, p 0.001; lower platelet...... adenosine diphosphate, thrombin receptor-activating peptide, arachidonic acid, collagen, and collagen-related peptide. Patients had lower median platelet count than healthy individuals, 125 × 10(9)/L (interquartile range [IQR] 90-185) versus 240 × 10(9) (IQR 204-285), p 0.001. Platelet activation levels...... activation capacity being associated with low platelet count and Child-Pugh class B/C cirrhosis. Flow cytometric platelet aggregation was reduced in patients for collagen-related peptide and for adenosine diphosphate, e.g., platelet aggregation (mean ± standard deviation) was 57% ± 4 in patients versus 70...

  7. Early Stent Thrombosis and Mortality After Primary Percutaneous Coronary Intervention in ST-Segment-Elevation Myocardial Infarction

    DEFF Research Database (Denmark)

    Dangas, George D; Schoos, Mikkel M.; Steg, Philippe Gabriel

    2016-01-01

    BACKGROUND: Early stent thrombosis (ST) within 30 days after primary percutaneous coronary intervention in ST-segment-elevation myocardial infarction is a serious event. We sought to determine the predictors of and risk of mortality after early ST according to procedural antithrombotic therapy...... percutaneous coronary intervention with stent implantation at 188 international sites, randomized to either bivalirudin or heparin±a glycoprotein IIb/IIIa inhibitor (GPI). Early ST occurred in 100 patients (2.0%), 20 of whom (20.0%) died. Bivalirudin was associated with higher rates of early ST compared...... with bivalirudin compared with heparin±GPI because of increased ST within 4 hours after primary percutaneous coronary intervention. However, the mortality attributable to early ST was significantly lower after bivalirudin than after heparin±GPI. CLINICAL TRIAL REGISTRATION: URL: http...

  8. Idiopathic thrombocytopenic purpura

    Directory of Open Access Journals (Sweden)

    L Kayal

    2014-01-01

    Full Text Available Idiopathic thrombocytopenic purpura (ITP is defined as a hematologic disorder, characterized by isolated thrombocytopenia without a clinically apparent cause. The major causes of accelerated platelet consumption include immune thrombocytopenia, decreased bone marrow production, and increased splenic sequestration. The clinical presentation may be acute with severe bleeding, or insidious with slow development with mild or no symptoms. The initial laboratory tests useful at the first visit to predict future diagnosis were erythrocyte count, leukocyte count, anti-glycoprotein IIb/IIIa antibodies, reticulated platelets, plasma thrombopoietin level. Treatment should be restricted to those patients with moderate or severe thrombocytopenia who are bleeding or at risk of bleeding. We present a case report on ITP with clinical presentation, diagnosis and management.

  9. Update on intravenous fibrinolytic therapy for acute myocardial infarction.

    Science.gov (United States)

    Wright, R S; Kopecky, S L; Reeder, G S

    2000-11-01

    Intravenous fibrinolytic therapy is used widely in the treatment of ST-elevation acute myocardial infarction. Advances in this therapeutic modality during the past 5 years include new third-generation fibrinolytic agents and creative strategies to enhance administration and efficacy of fibrinolytic therapy. Several of the new agents allow for single- or double-bolus injection. A number of ongoing large randomized trials are attempting to determine whether the combination of fibrinolytic therapy with low-molecular-weight heparin or a glycoprotein IIb/IIIa antagonist enhances coronary reperfusion and reduces mortality and late reocclusion. One large prospective trial is investigating the potential benefit of prehospital administration of fibrinolytic therapy. This article summarizes recent safety and efficacy data on fibrinolytic therapy, with particular emphasis on the new third-generation fibrin-specific agents; reviews the preliminary data on facilitated fibrinolysis; and discusses the rationale for prehospital administration of fibrinolytic therapy.

  10. CdTe quantum dots induce activation of human platelets: implications for nanoparticle hemocompatibility.

    Science.gov (United States)

    Samuel, Stephen P; Santos-Martinez, Maria J; Medina, Carlos; Jain, Namrata; Radomski, Marek W; Prina-Mello, Adriele; Volkov, Yuri

    2015-01-01

    New nanomaterials intended for systemic administration have raised concerns regarding their biocompatibility and hemocompatibility. Quantum dots (QD) nanoparticles have been used for diagnostics, and recent work suggests their use for in vivo molecular and cellular imaging. However, the hemocompatibility of QDs and their constituent components has not been fully elucidated. In the present study, comprehensive investigation of QD-platelet interactions is presented. These interactions were shown using transmission electron microscopy. The effects of QDs on platelet function were investigated using light aggregometry, quartz crystal microbalance with dissipation, flow cytometry, and gelatin zymography. Platelet morphology was also analyzed by phase-contrast, immunofluorescence, atomic-force and transmission electron microscopy. We show that the QDs bind to platelet plasma membrane with the resultant upregulation of glycoprotein IIb/IIIa and P-selectin receptors, and release of matrix metalloproteinase-2. These findings unravel for the first time the mechanism of functional response of platelets to ultrasmall QDs in vitro.

  11. Internalization and Axonal Transport of the HIV Glycoprotein gp120

    Science.gov (United States)

    Berth, Sarah; Caicedo, Hector Hugo; Sarma, Tulika; Morfini, Gerardo

    2015-01-01

    The HIV glycoprotein gp120, a neurotoxic HIV glycoprotein that is overproduced and shed by HIV-infected macrophages, is associated with neurological complications of HIV such as distal sensory polyneuropathy, but interactions of gp120 in the peripheral nervous system remain to be characterized. Here, we demonstrate internalization of extracellular gp120 in a manner partially independent of binding to its coreceptor CXCR4 by F11 neuroblastoma cells and cultured dorsal root ganglion neurons. Immunocytochemical and pharmacological experiments indicate that gp120 does not undergo trafficking through the endolysosomal pathway. Instead, gp120 is mainly internalized through lipid rafts in a cholesterol-dependent manner, with a minor fraction being internalized by fluid phase pinocytosis. Experiments using compartmentalized microfluidic chambers further indicate that, after internalization, endocytosed gp120 selectively undergoes retrograde but not anterograde axonal transport from axons to neuronal cell bodies. Collectively, these studies illuminate mechanisms of gp120 internalization and axonal transport in peripheral nervous system neurons, providing a novel framework for mechanisms for gp120 neurotoxicity. PMID:25636314

  12. Enhancing comparative rabies DNA vaccine effectiveness through glycoprotein gene modifications.

    Science.gov (United States)

    Osinubi, M O V; Wu, X; Franka, R; Niezgoda, M; Nok, A J; Ogunkoya, A B; Rupprecht, C E

    2009-11-27

    Enhancing DNA vaccine effectiveness remains a challenge, especially if the desired goal is immunization efficacy after a single dose. The glycoprotein gene from the rabies virus Evelyn-Rokitnicki-Abelseth (ERA) strain was modified by mutation at amino acid residue 333 from arginine to glutamine. The modified and original unmodified glycoprotein genes were cloned separately and developed as DNA vaccines for immunization in mice. The intramuscular (IM) route using a single dose (100 microg) of a modified DNA vaccine showed virus neutralizing antibody induction by d30, and 80% of the mice survived a challenge in which 100% of unvaccinated controls succumbed. Similar results were obtained using a single dose (10 microg) by the intradermal (ID) route with one-tenth amount of the DNA administered. Administration of single dose of DNA vaccine with unmodified G did not result in the production of detectable levels of virus neutralizing antibody by d30. The results of the IM and the ID routes of administration were statistically significant (Prabies virus strain may be an ideal candidate for DNA vaccine efficacy enhancement.

  13. Glycoprotein NMB: an Emerging Role in Neurodegenerative Disease.

    Science.gov (United States)

    Budge, Kevin M; Neal, Matthew L; Richardson, Jason R; Safadi, Fayez F

    2017-08-30

    Neurodegeneration is characterized by severe neuronal loss leading to the cognitive and physical impairments that define various neurodegenerative diseases. Neuroinflammation is one hallmark of neurodegenerative diseases and can ultimately contribute to disease progression. Increased inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1β (IL-1 β), and tumor necrosis factor-α (TNF-α) are associated with Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Unfortunately, current therapeutic options lack ability to stop or effectively slow progression of these diseases and are primarily aimed at alleviating symptoms. Thus, it is crucial to discover novel treatment candidates for neurodegenerative diseases. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type-I transmembrane glycoprotein first identified in a melanoma cell line. GPNMB augments bone mineral deposition by stimulating osteoblast differentiation. Aside from its anabolic function in the bone, emerging evidence suggests that GPNMB has anti-inflammatory and reparative functions. GPNMB has also been demonstrated to be neuroprotective in an animal model of ALS, cerebral ischemia, and other disease models. Given these discoveries, GPNMB should be investigated as a potential therapeutic option for multiple neurodegenerative diseases.

  14. In vivo RNA interference-mediated ablation of MDR1 P-glycoprotein

    NARCIS (Netherlands)

    Pichler, Andrea; Zelcer, Noam; Prior, Julie L.; Kuil, Annemieke J.; Piwnica-Worms, David

    2005-01-01

    Multidrug resistance (MDR) remains a major obstacle to successful chemotherapeutic treatment of cancer and can be caused by overexpression of P-glycoprotein, the MDR1 gene product. To further validate a knockdown approach for circumventing MDR, we developed a P-glycoprotein inhibition strategy using

  15. Systemic alteration of cell-surface and secreted glycoprotein expression in malignant breast cancer cell lines.

    Science.gov (United States)

    Timpe, Leslie C; Yen, Roger; Haste, Nicole V; Litsakos-Cheung, Christina; Yen, Ten-Yang; Macher, Bruce A

    2013-11-01

    Breast cancer cell lines express fewer transmembrane and secreted glycoproteins than nonmalignant ones. The objective of these experiments was to characterize the changes in the expression of several hundred glycoproteins quantitatively. Secreted and cell-surface glycoproteins were isolated using a glycoprotein capture protocol and then identified by tandem mass spectrometry. Glycoproteins expressed by a group of cell lines originating from malignant tumors of the breast were compared with those expressed by a nonmalignant set. The average number of spectral counts (proportional to relative protein abundance) and the total number of glycopeptides in the malignant samples were reduced to about two-thirds of the level in the nonmalignant samples. Most glycoproteins were expressed at a different level in the malignant samples, with nearly as many increasing as decreasing. The glycoproteins with reduced expression accounted for a larger change in spectral counts, and hence for the net loss of spectral counts in the malignant lines. Similar results were found when the glycoproteins were studied via identified glycosylation sites only, or through identified sites together with non-glycopeptides. The overall reduction is largely due to the loss of integrins, laminins and other proteins that form or interact with the basement membrane.

  16. Glycoproteins of mouse vaginal epithelium: differential expression related to estrous cyclicity

    DEFF Research Database (Denmark)

    Horvat, B; Multhaupt, H A; Damjanov, I

    1993-01-01

    We used lectin overlay blotting and SDS-PAGE to analyze the estrous cycle-specific expression of mouse vaginal epithelial glycoproteins. Seven lectins chosen for their differential carbohydrate-binding specificity revealed 15 glycoproteins that showed cycle-related expression. Each lectin had...

  17. Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants

    NARCIS (Netherlands)

    Wilbers, Ruud H. P.; Westerhof, Lotte B.; van Noort, Kim; Obieglo, Katja; Driessen, Nicole N.; Everts, Bart; Gringhuis, Sonja I.; Schramm, Gabriele; Goverse, Aska; Smant, Geert; Bakker, Jaap; Smits, Hermelijn H.; Yazdanbakhsh, Maria; Schots, Arjen; Hokke, Cornelis H.

    2017-01-01

    Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are

  18. Interaction of human erythrocyte MN glycoprotein with rabbit IgG immunoglobulins.

    Science.gov (United States)

    Białkowska, H; Morawiecki, A

    1978-01-01

    The interaction of rabbit non-specific IgG and human erythrocyte glycoprotein was investigated using the solvent perturbation difference spectroscopy method. This interaction manifested itself by decreasing accessibility of chromophores to perturbants. Masking of the chromophores was abolished by low detergent concentrations and by changes of native IgG structure by 3 M urea. The sialic acid residues of the glycoprotein were necessary for this effect but probably not due to simple electrostatic interactions. It seems that the IgG-glycoprotein interaction requires intact both--the IgG molecule structure and the structure of the glycoprotein micelle. Interaction of this kind was not observed between glycoprotein and some other proteins as bovine serum albumin, alpha-chymotrypsynogen and human IgA.

  19. Leukocyte count is a modulating factor for the mortality benefit of bivalirudin in ST-segment-elevation acute myocardial infarction: the HORIZONS-AMI trial.

    Science.gov (United States)

    Palmerini, Tullio; Brener, Sorin J; Mehran, Roxana; Dangas, George; Genereux, Philippe; Riva, Diego Della; Mariani, Andrea; Xu, Ke; Stone, Gregg W

    2013-10-01

    Although the reduction in mortality with bivalirudin compared with unfractionated heparin plus glycoprotein IIb/IIIa inhibitors in the Harmonizing Outcome with Revascularization and Stent in Acute Myocardial Infarction (HORIZONS-AMI) trial has been attributed to lower rates of major bleeding, alternative mechanisms have not been investigated in depth. We sought to investigate whether there might be an interaction between white blood cell (WBC) count and bivalirudin for the risk of mortality, and whether this interaction is independent of major bleeding. Among the 3602 patients enrolled in the HORIZONS-AMI trial, WBC count was available in 3433 (95.3%) patients. Patients were stratified according to WBC tertiles. At 1-year follow-up, bivalirudin was associated with significantly lower rates of mortality and cardiac mortality compared with unfractionated heparin plus glycoprotein IIb/IIIa inhibitors in patients in the upper WBC tertile (all-cause death: 4.1% versus 9.3%, respectively; P=0.0004; cardiac death: 2.0% versus 6.9%; respectively; P<0.0001) but not in patients in the mid-WBC or lower WBC tertiles. The reduction of mortality with bivalirudin across WBC tertiles was independent of major bleeding, and a significant interaction was apparent for 1-year all-cause mortality and cardiac mortality between WBC and bivalirudin therapy. Similar findings were apparent at 3 years. In patients with ST-segment-elevation myocardial infarction, a significant interaction between bivalirudin therapy and admission WBC count was apparent for 1-year mortality. The reduction in mortality was independent of major bleeding, suggesting that other mechanisms may be implicated in the survival benefit observed with bivalirudin. http://www.clinicaltrials.gov. Unique identifier: NCT00433966.

  20. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression

    Science.gov (United States)

    Prados, Jose; Caba, Octavio; Cabeza, Laura; Berdasco, Maria; Gónzalez, Beatriz; Melguizo, Consolación

    2015-01-01

    Background The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated. Methods Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2’-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed. Results Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines. Conclusions These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma

  1. Comparison of α1-Antitrypsin, α1-Acid Glycoprotein, Fibrinogen and NOx as Indicator of Subclinical Mastitis in Riverine Buffalo (

    Directory of Open Access Journals (Sweden)

    Anirban Guha

    2013-06-01

    Full Text Available Mastitis set apart as clinical and sub clinical is a disease complex of dairy cattle, with sub clinical being the most important economically. Of late, laboratories showed interest in developing biochemical markers to diagnose sub clinical mastitis (SCM in herds. Many workers reported noteworthy alternation of acute phase proteins (APPs and nitric oxide, (measured as nitrate+nitrite = NOx in milk due to intra-mammary inflammation. But, the literature on validation of these parameters as indicators of SCM, particularly in riverine milch buffalo (Bubalus bubalis milk is inadequate. Hence, the present study focused on comparing several APPs viz. α1- anti trypsin, α1- acid glycoprotein, fibrinogen and NOx as indicators of SCM in buffalo milk. These components in milk were estimated using standardized analytical protocols. Somatic cell count (SCC was done microscopically. Microbial culture was done on 5% ovine blood agar. Of the 776 buffaloes (3,096 quarters sampled, only 347 buffaloes comprising 496 quarters were found positive for SCM i.e. milk culture showed growth in blood agar with SCC≥2×105 cells/ml of milk. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. It was observed that α1- anti trypsin and NOx had a highly significant (p<0.01 increase in SCM milk, whereas, the increase of α1- acid glycoprotein in infected milk was significant (p<0.05. Fibrinogen was below detection level in both healthy and SCM milk. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×105 cells/ml of milk as the benchmark. Udder profile correlation coefficient was also used. Allowing for statistical and epidemiological analysis, it was concluded that α1- anti trypsin indicates SCM irrespective of etiology, whereas α1- acid glycoprotein better diagnosed SCM caused by gram positive bacteria. NOx did not prove to

  2. Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Leopold; Giang, Erick; Nieusma, Travis; Kadam, Rameshwar U.; Cogburn, Kristin E.; Hua, Yuanzi; Dai, Xiaoping; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Law, Mansun

    2014-08-26

    Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design.

  3. Human Milk Glycoproteins Protect Infants Against Human Pathogens

    Science.gov (United States)

    Liu, Bo

    2013-01-01

    Abstract Breastfeeding protects the neonate against pathogen infection. Major mechanisms of protection include human milk glycoconjugates functioning as soluble receptor mimetics that inhibit pathogen binding to the mucosal cell surface, prebiotic stimulation of gut colonization by favorable microbiota, immunomodulation, and as a substrate for bacterial fermentation products in the gut. Human milk proteins are predominantly glycosylated, and some biological functions of these human milk glycoproteins (HMGPs) have been reported. HMGPs range in size from 14 kDa to 2,000 kDa and include mucins, secretory immunoglobulin A, bile salt-stimulated lipase, lactoferrin, butyrophilin, lactadherin, leptin, and adiponectin. This review summarizes known biological roles of HMGPs that may contribute to the ability of human milk to protect neonates from disease. PMID:23697737

  4. Immunoglobulin-E reactivity to wine glycoproteins in heavy drinkers

    DEFF Research Database (Denmark)

    Gonzalez-Quintela, Arturo; Gomez-Rial, Jose; Valcarcel, Catalina

    2011-01-01

    and biological significance of IgE antibodies to N-glycans from wine glycoproteins in heavy drinkers. A structured questionnaire, skin prick tests, serum IgE levels, IgE-immunoblotting to wine extracts, and basophil activation tests were used to characterize 20 heavy drinkers and 10 control subjects. Eleven...... heavy drinkers (55%) showed IgE binding to proteins in wine extracts. The proteins were identified by mass spectrometry as grape-derived vacuolar invertase and thaumatin-like protein. Immunoblot reactivity was closely associated with the presence of IgE to CCDs and was inhibited by preincubation...... with a glycoconjugate containing bromelain-type N-glycans. The same conjugate, CCD-bearing allergens, and wine extracts activated basophils in patients with high-titer CCD-specific IgE but not in healthy controls. There was no relationship between immunoblot reactivity and consumption of any specific type of wine...

  5. Small-angle scattering study of Aspergillus awamori glycoprotein glucoamylase

    International Nuclear Information System (INIS)

    Schmidt, A. E.; Shvetsov, A. V.; Kuklin, A. I.; Lebedev, D. V.; Surzhik, M. A.; Sergeev, V. R.; Isaev-Ivanov, V. V.

    2016-01-01

    Glucoamylase from fungus Aspergillus awamori is glycoside hydrolase that catalyzes the hydrolysis of α-1,4- and α-1,6-glucosidic bonds in glucose polymers and oligomers. This glycoprotein consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated polypeptide chain. The conformation of the linker, the relative arrangement of the domains, and the structure of the full-length enzyme are unknown. The structure of the recombinant glucoamylase GA1 was studied by molecular modelling and small-angle neutron scattering (SANS) methods. The experimental SANS data provide evidence that glucoamylase exists as a monomer in solution and contains a glycoside component, which makes a substantial contribution to the scattering. The model of full-length glucoamylase, which was calculated without taking into account the effect of glycosylation, is consistent with the experimental data and has a radius of gyration of 33.4 ± 0.6 Å

  6. Small-angle scattering study of Aspergillus awamori glycoprotein glucoamylase

    Energy Technology Data Exchange (ETDEWEB)

    Schmidt, A. E., E-mail: schmidt@omrb.pnpi.spb.ru; Shvetsov, A. V. [National Research Center “Kurchatov Institute”, Konstantinov Petersburg Nuclear Physics Institute (Russian Federation); Kuklin, A. I. [Joint Institute for Nuclear Research (Russian Federation); Lebedev, D. V.; Surzhik, M. A.; Sergeev, V. R.; Isaev-Ivanov, V. V. [National Research Center “Kurchatov Institute”, Konstantinov Petersburg Nuclear Physics Institute (Russian Federation)

    2016-01-15

    Glucoamylase from fungus Aspergillus awamori is glycoside hydrolase that catalyzes the hydrolysis of α-1,4- and α-1,6-glucosidic bonds in glucose polymers and oligomers. This glycoprotein consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated polypeptide chain. The conformation of the linker, the relative arrangement of the domains, and the structure of the full-length enzyme are unknown. The structure of the recombinant glucoamylase GA1 was studied by molecular modelling and small-angle neutron scattering (SANS) methods. The experimental SANS data provide evidence that glucoamylase exists as a monomer in solution and contains a glycoside component, which makes a substantial contribution to the scattering. The model of full-length glucoamylase, which was calculated without taking into account the effect of glycosylation, is consistent with the experimental data and has a radius of gyration of 33.4 ± 0.6 Å.

  7. Small-angle scattering study of Aspergillus awamori glycoprotein glucoamylase

    Science.gov (United States)

    Schmidt, A. E.; Shvetsov, A. V.; Kuklin, A. I.; Lebedev, D. V.; Surzhik, M. A.; Sergeev, V. R.; Isaev-Ivanov, V. V.

    2016-01-01

    Glucoamylase from fungus Aspergillus awamori is glycoside hydrolase that catalyzes the hydrolysis of α-1,4- and α-1,6-glucosidic bonds in glucose polymers and oligomers. This glycoprotein consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated polypeptide chain. The conformation of the linker, the relative arrangement of the domains, and the structure of the full-length enzyme are unknown. The structure of the recombinant glucoamylase GA1 was studied by molecular modelling and small-angle neutron scattering (SANS) methods. The experimental SANS data provide evidence that glucoamylase exists as a monomer in solution and contains a glycoside component, which makes a substantial contribution to the scattering. The model of full-length glucoamylase, which was calculated without taking into account the effect of glycosylation, is consistent with the experimental data and has a radius of gyration of 33.4 ± 0.6 Å.

  8. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    Science.gov (United States)

    Xu, Kai; Rockx, Barry; Xie, Yihu; DeBuysscher, Blair L; Fusco, Deborah L; Zhu, Zhongyu; Chan, Yee-Peng; Xu, Yan; Luu, Truong; Cer, Regina Z; Feldmann, Heinz; Mokashi, Vishwesh; Dimitrov, Dimiter S; Bishop-Lilly, Kimberly A; Broder, Christopher C; Nikolov, Dimitar B

    2013-01-01

    The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

  9. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Kai Xu

    Full Text Available The henipaviruses, represented by Hendra (HeV and Nipah (NiV viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4 was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

  10. Human adenovirus 52 uses sialic acid-containing glycoproteins and the coxsackie and adenovirus receptor for binding to target cells.

    Science.gov (United States)

    Lenman, Annasara; Liaci, A Manuel; Liu, Yan; Årdahl, Carin; Rajan, Anandi; Nilsson, Emma; Bradford, Will; Kaeshammer, Lisa; Jones, Morris S; Frängsmyr, Lars; Feizi, Ten; Stehle, Thilo; Arnberg, Niklas

    2015-02-01

    Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR) protein. Human adenovirus type 52 (HAdV-52) is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK) binds to CAR, and the knob domain of the short fiber (52SFK) binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy.

  11. Human adenovirus 52 uses sialic acid-containing glycoproteins and the coxsackie and adenovirus receptor for binding to target cells.

    Directory of Open Access Journals (Sweden)

    Annasara Lenman

    2015-02-01

    Full Text Available Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR protein. Human adenovirus type 52 (HAdV-52 is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK binds to CAR, and the knob domain of the short fiber (52SFK binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy.

  12. Platelet activation in patients with the Raynaud phenomenon.

    Science.gov (United States)

    Polidoro, L; Barnabei, R; Giorgini, P; Petrazzi, L; Ferri, C; Properzi, G

    2012-05-01

    The Raynaud phenomenon (RP) is an exaggerated and reversible vasospasm of small arteries triggered by cold or emotional stress. Primary RP (PRP) term is used when the underlying condition is unknown. An altered regulation in vascular tone and/or release of soluble mediators from activated platelets plays a role in PRP through an increased oxidative stress. We assessed platelet activation and oxidative stress in patients with PRP by measuring platelet PAC-1, an index of glycoprotein (Gp) IIb/IIIa receptor activation, thromboxane A(2) (TXA(2)), an index of platelet activation and 8-epi-prostaglandin F(2α) (8-epi-PGF(2α)), a marker of endogenous in vivo peroxidation. Eighteen asymptomatic patients with PRP (age 41.37 ± 16.94 years; 17 women, 1 man) and 18 healthy subjects (age of 35.11 ± 13.16 years; 16 women, 2 men) were studied. PAC-1 was analysed by flow cytometry while circulating TXB(2) , a stable metabolite of TXA(2) and 8-epi-PGF(2α) levels were assessed by ELISA kit. Our results show a significant platelet activation in PRP patients as indicated by increased PAC-1 expression (65.29 ± 15.24%; P < 0.001), TXB(2) (1477.83 ± 454.04 pg/mL; P= 0.003) and 8-epi-PGF(2α) circulating levels (42.50 ± 14.14 ng/mL; P < 0.001). An inverse correlation between the degree of PAC-1 expression and TXB(2) levels (r=-0.527; P= 0.02) was also found in PRP patients, suggesting that downregulation of GpIIb/IIIa receptor expression may occur during thrombocytopoiesis, as a consequence of the chronic exposure to increased TXB(2) concentration. Our study for the first time shows a marked activation of GpIIb/IIIa receptor in asymptomatic patients with PRP and supports antiplatelet therapy in PRP patients. © 2010 The Authors. Internal Medicine Journal © 2010 Royal Australasian College of Physicians.

  13. Crystal Structure of the Human Cytomegalovirus Glycoprotein B.

    Directory of Open Access Journals (Sweden)

    Heidi G Burke

    2015-10-01

    Full Text Available Human cytomegalovirus (HCMV, a dsDNA, enveloped virus, is a ubiquitous pathogen that establishes lifelong latent infections and caused disease in persons with compromised immune systems, e.g., organ transplant recipients or AIDS patients. HCMV is also a leading cause of congenital viral infections in newborns. Entry of HCMV into cells requires the conserved glycoprotein B (gB, thought to function as a fusogen and reported to bind signaling receptors. gB also elicits a strong immune response in humans and induces the production of neutralizing antibodies although most anti-gB Abs are non-neutralizing. Here, we report the crystal structure of the HCMV gB ectodomain determined to 3.6-Å resolution, which is the first atomic-level structure of any betaherpesvirus glycoprotein. The structure of HCMV gB resembles the postfusion structures of HSV-1 and EBV homologs, establishing it as a new member of the class III viral fusogens. Despite structural similarities, each gB has a unique domain arrangement, demonstrating structural plasticity of gB that may accommodate virus-specific functional requirements. The structure illustrates how extensive glycosylation of the gB ectodomain influences antibody recognition. Antigenic sites that elicit neutralizing antibodies are more heavily glycosylated than those that elicit non-neutralizing antibodies, which suggest that HCMV gB uses glycans to shield neutralizing epitopes while exposing non-neutralizing epitopes. This glycosylation pattern may have evolved to direct the immune response towards generation of non-neutralizing antibodies thus helping HCMV to avoid clearance. HCMV gB structure provides a starting point for elucidation of its antigenic and immunogenic properties and aid in the design of recombinant vaccines and monoclonal antibody therapies.

  14. In silico-based vaccine design against Ebola virus glycoprotein

    Directory of Open Access Journals (Sweden)

    Dash R

    2017-03-01

    Full Text Available Raju Dash,1 Rasel Das,2 Md Junaid,3 Md Forhad Chowdhury Akash,4 Ashekul Islam,5 SM Zahid Hosen1 1Molecular Modeling and Drug Design Laboratory (MMDDL, Pharmacology Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR, Chittagong, Bangladesh; 2Nanotechnology and Catalysis Research Center, University of Malaya, Kuala Lumpur, Malaysia; 3Department of Pharmaceutical Sciences, North South University, Dhaka, Bangladesh; 4Department of Pharmacy, BGC Trust University Bangladesh, Chittagong, Bangladesh; 5Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong, Bangladesh Abstract: Ebola virus (EBOV is one of the lethal viruses, causing more than 24 epidemic outbreaks to date. Despite having available molecular knowledge of this virus, no definite vaccine or other remedial agents have been developed yet for the management and avoidance of EBOV infections in humans. Disclosing this, the present study described an epitope-based peptide vaccine against EBOV, using a combination of B-cell and T-cell epitope predictions, followed by molecular docking and molecular dynamics simulation approach. Here, protein sequences of all glycoproteins of EBOV were collected and examined via in silico methods to determine the most immunogenic protein. From the identified antigenic protein, the peptide region ranging from 186 to 220 and the sequence HKEGAFFLY from the positions of 154–162 were considered the most potential B-cell and T-cell epitopes, correspondingly. Moreover, this peptide (HKEGAFFLY interacted with HLA-A*32:15 with the highest binding energy and stability, and also a good conservancy of 83.85% with maximum population coverage. The results imply that the designed epitopes could manifest vigorous enduring defensive immunity against EBOV. Keywords: Ebola virus, epitope, glycoprotein, vaccine design

  15. Factors affecting recombinant Western equine encephalitis virus glycoprotein production in the baculovirus system.

    Science.gov (United States)

    Toth, Ann M; Geisler, Christoph; Aumiller, Jared J; Jarvis, Donald L

    2011-12-01

    In an effort to produce processed, soluble Western equine encephalitis virus (WEEV) glycoproteins for subunit therapeutic vaccine studies, we isolated twelve recombinant baculoviruses designed to express four different WEEV glycoprotein constructs under the transcriptional control of three temporally distinct baculovirus promoters. The WEEV glycoprotein constructs encoded full-length E1, the E1 ectodomain, an E26KE1 polyprotein precursor, and an artificial, secretable E2E1 chimera. The three different promoters induced gene expression during the immediate early (ie1), late (p6.9), and very late (polh) phases of baculovirus infection. Protein expression studies showed that the nature of the WEEV construct and the timing of expression both influenced the quantity and quality of recombinant glycoprotein produced. The full-length E1 product was insoluble, irrespective of the timing of expression. Each of the other three constructs yielded soluble products and, in these cases, the timing of expression was important, as higher protein processing efficiencies were generally obtained at earlier times of infection. However, immediate early expression did not yield detectable levels of every WEEV product, and expression during the late (p6.9) or very late (polh) phases of infection provided equal or higher amounts of processed, soluble product. Thus, while earlier foreign gene expression can provide higher recombinant glycoprotein processing efficiencies in the baculovirus system, in the case of the WEEV glycoproteins, earlier expression did not provide larger amounts of high quality, soluble recombinant glycoprotein product. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. THE ROLE OF P-GLYCOPROTEIN IN RATIONAL PHARMACOTHERAPY IN CARDIOLOGY

    Directory of Open Access Journals (Sweden)

    A. V. Shulkin

    2015-09-01

    Full Text Available On the basis of the analysis of published data the role of P-glycoprotein, carrier protein, in rational pharmacotherapy in cardiology was shown on the example of its substrates – digoxin, antiplatelet agents and anticoagulants. Determination of C3435T polymorphism of multidrug resistance gene (MDR1, encoding P-glycoprotein, in pharmacotherapy with digoxin, antiplatelet drugs (clopidogrel tikagrelol, prasugrel and anticoagulants (dabigatran etexilate, rivaroxaban, edoxaban is not feasible in routine practice. Drug in- teractions have clinical implications for the efficacy and safety of pharmacotherapy in coadministration of these drugs with P-glycoprotein substrates, inducers and inhibitors.

  17. THE ROLE OF P-GLYCOPROTEIN IN RATIONAL PHARMACOTHERAPY IN CARDIOLOGY

    Directory of Open Access Journals (Sweden)

    A. V. Shulkin

    2013-01-01

    Full Text Available On the basis of the analysis of published data the role of P-glycoprotein, carrier protein, in rational pharmacotherapy in cardiology was shown on the example of its substrates – digoxin, antiplatelet agents and anticoagulants. Determination of C3435T polymorphism of multidrug resistance gene (MDR1, encoding P-glycoprotein, in pharmacotherapy with digoxin, antiplatelet drugs (clopidogrel tikagrelol, prasugrel and anticoagulants (dabigatran etexilate, rivaroxaban, edoxaban is not feasible in routine practice. Drug in- teractions have clinical implications for the efficacy and safety of pharmacotherapy in coadministration of these drugs with P-glycoprotein substrates, inducers and inhibitors.

  18. Release of cell coat glycoproteins from the human blood lymphocytes after UV irradiation (254 nm)

    Energy Technology Data Exchange (ETDEWEB)

    Artsishevskaya, R.A.; Mironova, A.P.; Samojlova, K.A. (AN SSSR, Leningrad. Inst. Tsitologii)

    1984-01-01

    Irradiation of the human peripheric blood lymphocytes by UV rays (254 nm) in nonlethal doses is accompanied by the decrease (8-13%) of sorption by them of man's life time of alcyane blue dya which selectively is bound by glycoproteins, glycolipides and acid mucopolysaccharides of cellular surface. As simultaneously the yield from substance cells by some properties similar to glycoproteins is intensified by 9-15%, an assumption is made that from the surface of UV-irradiated lymphocites glycoproteins are disorbed. This effect is discussed in connection with possible primary mechanisms of medical-sanitation effect of UV irradiation.

  19. A glycoprotein from Porphyra yezoensis produces anti-inflammatory effects in liposaccharide-stimulated macrophages via the TLR4 signaling pathway.

    Science.gov (United States)

    Shin, Eun-Soon; Hwang, Hye-Jung; Kim, In-Hye; Nam, Taek-Jeong

    2011-11-01

    The purpose of this study was to investigate the antioxidant and anti-inflammatory effects of a glycoprotein isolated from the alga Porphyra yezoensis in LPS-stimulated RAW 264.7 mouse macrophages. First, we extracted a novel material with antioxidant activity from P. yezoensis, confirmed by SDS-PAGE to be a glycoprotein, which we named P. yezoensis glycoprotein (PGP). PGP inhibited the production of NO and ROS and expression of iNOS, COX-2, TNF-α and IL-1β, which are involved in the pathogenesis of many inflammation-associated human diseases, including septic shock, hemorrhagic shock and rheumatoid arthritis. Next, we determined the mechanisms behind the antioxidant and anti-inflammatory activities of PGP. We focused on the Toll-like receptor 4 (TLR4) signaling pathway because it is well-known to induce the pro-inflammatory proteins that trigger MAPK and NF-κB activation in lipopolysaccharide (LPS)-induced oxidative events. PGP treatment reduced the formation of the TLR4-IRAK4 and TLR4-TRIF binding complexes in response to LPS. Moreover, it inhibited LPS-induced activation and nuclear translocation of NF-κB by abrogating IκB phosphorylation. PGP also suppressed the phosphorylation of ERK1/2 and JNK in a dose-dependent manner. These results suggest that PGP exerts its anti-inflammatory effects by modulating TLR4 signaling and thus inhibiting the activation of NF-κB and MAP kinases.

  20. Management strategies in antithrombotic therapy

    National Research Council Canada - National Science Library

    Lincoff, A. Michael; Messerli, Adrian W; Askari, Arman

    2007-01-01

    ... in the management of non-st-elevation ACS 4.5 GP IIb/IIIa inhibitors in the management of acute STEMI 4.6 Safety of GP IIb/IIIa inhibitors 4.7 Summary 77 77 78 80 87 91 94 96 Chapter 5 Unfractionated Hepa...

  1. Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method.

    Science.gov (United States)

    Kamoda, Satoru; Nakanishi, Yasuharu; Kinoshita, Mitsuhiro; Ishikawa, Rika; Kakehi, Kazuaki

    2006-02-17

    Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved 60-360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of alpha1-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower pI values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher pI values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides.

  2. Nonsense mutation in the glycoprotein Ibα coding sequence associated with Bernard-Soulier syndrome

    International Nuclear Information System (INIS)

    Ware, J.; Russell, S.R.; Vicente, V.; Scharf, R.E.; Tomer, A.; McMillian, R.; Ruggeri, Z.M.

    1990-01-01

    Three distinct gene products, the α and β chains of glycoprotein (GP) Ib and GP IX, constitute the platelet membrane GP Ib-IX complex, a receptor for von Willebrand factor and thrombin involved in platelet adhesion and aggregation. Defective function of the GP Ib-IX complex is the hallmark of a rare congenital bleeding disorder of still undefined pathogenesis, the Bernard-Soulier syndrome. The authors have analyzed the molecular basis of the disease in one patient in whom immunoblotting of solubilized platelets demonstrated absence of normal GP Ibα but presence of a smaller immunoreactive species. The truncated polypeptide was also present, along with normal protein, in platelets from the patient's mother and two of his four children. Genetic characterization identified a nucleotide transition changing the Trp-343 codon (TGG) to a nonsense codon (TGA). Such a mutation explains the origin of the smaller GP Ibα, which by lacking half of the sequence on the carboxyl-terminal side, including the transmembrane domain, cannot be properly inserted in the platelet membrane. Both normal and mutant codons were found in the patient, suggesting that he is a compound heterozygote with a still unidentified defect in the other GP Ibα allele. Nonsense mutation and truncated GP Ibα polypeptide were found to cosegregate in four individuals through three generations and were associated with either Bernard-Soulier syndrome or carrier state phenotype. The molecular abnormality demonstrated in this family provides evidence that defective synthesis of GP Ibα alters the membrane expression of the GP Ib-IX complex and may be responsible for Bernard-Soulier syndrome

  3. MAGIC: an automated N-linked glycoprotein identification tool using a Y1-ion pattern matching algorithm and in silico MS² approach.

    Science.gov (United States)

    Lynn, Ke-Shiuan; Chen, Chen-Chun; Lih, T Mamie; Cheng, Cheng-Wei; Su, Wan-Chih; Chang, Chun-Hao; Cheng, Chia-Ying; Hsu, Wen-Lian; Chen, Yu-Ju; Sung, Ting-Yi

    2015-02-17

    Glycosylation is a highly complex modification influencing the functions and activities of proteins. Interpretation of intact glycopeptide spectra is crucial but challenging. In this paper, we present a mass spectrometry-based automated glycopeptide identification platform (MAGIC) to identify peptide sequences and glycan compositions directly from intact N-linked glycopeptide collision-induced-dissociation spectra. The identification of the Y1 (peptideY0 + GlcNAc) ion is critical for the correct analysis of unknown glycoproteins, especially without prior knowledge of the proteins and glycans present in the sample. To ensure accurate Y1-ion assignment, we propose a novel algorithm called Trident that detects a triplet pattern corresponding to [Y0, Y1, Y2] or [Y0-NH3, Y0, Y1] from the fragmentation of the common trimannosyl core of N-linked glycopeptides. To facilitate the subsequent peptide sequence identification by common database search engines, MAGIC generates in silico spectra by overwriting the original precursor with the naked peptide m/z and removing all of the glycan-related ions. Finally, MAGIC computes the glycan compositions and ranks them. For the model glycoprotein horseradish peroxidase (HRP) and a 5-glycoprotein mixture, a 2- to 31-fold increase in the relative intensities of the peptide fragments was achieved, which led to the identification of 7 tryptic glycopeptides from HRP and 16 glycopeptides from the mixture via Mascot. In the HeLa cell proteome data set, MAGIC processed over a thousand MS(2) spectra in 3 min on a PC and reported 36 glycopeptides from 26 glycoproteins. Finally, a remarkable false discovery rate of 0 was achieved on the N-glycosylation-free Escherichia coli data set. MAGIC is available at http://ms.iis.sinica.edu.tw/COmics/Software_MAGIC.html .

  4. The influence of N-linked glycosylation on the function of platelet glycoprotein VI.

    Science.gov (United States)

    Kunicki, Thomas J; Cheli, Yann; Moroi, Masaaki; Furihata, Kenichi

    2005-10-15

    Using recombinant human glycoprotein VI (GPVI), we evaluated the effect of N-linked glycosylation at the consensus site Asparagine92-Glycine-Serine94 (N92GS94) on binding of this platelet-specific receptor to its ligands, human type I collagen, collagen-related peptide (CRP), and the snake venom C-type lectin convulxin (CVX). In COS-7 cells transiently transfected with GPVI, deglycosylation with peptide-N-glycosidase F (PNGase F; specific for complex N-linked glycans) or tunicamycin decreases the molecular weight of GPVI and reduces transfected COS-7 cell binding to both CRP and CVX. In stably transfected Dami cells, the substitutions N92A or S94A, but not L95H, resulted in a 30% to 40% decrease in adhesion to CVX, but a 90% or greater decrease in adhesion to CRP and a 65% to 70% decrease in adhesion to type I collagen. Treatment with PNGase F, but not Endoglycosidase H (Endo H) (specific for high-mannose N-linked glycans), produced an equivalent decrease in molecular weight. Neither N92A nor S94A affected the expression of GPVI, based on the direct binding of murine anti-human GPVI monoclonal antibody 204-11 to transfected Dami cells. These findings indicate that N-linked glycosylation at N92 in human GPVI is not required for surface expression, but contributes to maximal adhesion to type I collagen, CRP and, to a lesser extent, CVX.

  5. Glycoprotein folding and quality-control mechanisms in protein-folding diseases

    Directory of Open Access Journals (Sweden)

    Sean P. Ferris

    2014-03-01

    Full Text Available Biosynthesis of proteins – from translation to folding to export – encompasses a complex set of events that are exquisitely regulated and scrutinized to ensure the functional quality of the end products. Cells have evolved to capitalize on multiple post-translational modifications in addition to primary structure to indicate the folding status of nascent polypeptides to the chaperones and other proteins that assist in their folding and export. These modifications can also, in the case of irreversibly misfolded candidates, signal the need for dislocation and degradation. The current Review focuses on the glycoprotein quality-control (GQC system that utilizes protein N-glycosylation and N-glycan trimming to direct nascent glycopolypeptides through the folding, export and dislocation pathways in the endoplasmic reticulum (ER. A diverse set of pathological conditions rooted in defective as well as over-vigilant ER quality-control systems have been identified, underlining its importance in human health and disease. We describe the GQC pathways and highlight disease and animal models that have been instrumental in clarifying our current understanding of these processes.

  6. Qualitative and Quantitative Analysis of Carbohydrate Modification on Glycoproteins from Seeds of Ginkgo biloba.

    Science.gov (United States)

    Wang, Ting; Hu, Xiao-Chun; Cai, Zhi-Peng; Voglmeir, Josef; Liu, Li

    2017-09-06

    Recent progress in the relationship between carbohydrate cross-reactive determinants (CCDs) and allergic response highlights the importance of carbohydrate moieties in the innate immune system. Previous research pointed out that the protein allergen in Ginkgo biloba seeds is glycosylated, and the oligosaccharides conjugated to these proteins might also contribute to the allergy. The aim of this study was to analyze carbohydrate moieties, especially N-linked glycans, of glycoproteins from Ginkgo seeds originating from different places for detailed structures, to enable further research on the role played by N-glycans in Ginkgo-caused allergy. Results of monosaccharide composition and immunoblotting assays indicated the existence of N-glycans. Detailed structural elucidation of the N-glycans was further carried out by means of hydrophilic interaction ultraperformance liquid chromatography (HILIC-UPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 14 out of 16 structures detected by UPLC were confirmed by MALDI-TOF-MS and tandem mass spectrometry, among which complex-type N-glycans bearing Lewis A determinants and high-mannose-type N-glycans were identified from Ginkgo seeds for the first time. Precise quantification of N-glycans was performed by use of an external standard, and both the absolute amount of each N-glycan and the percentage of different types of N-glycan showed significant diversity among the samples without any pattern of geographic variation.

  7. Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope.

    Science.gov (United States)

    Wu, Yan; Zhu, Yaohua; Gao, Feng; Jiao, Yongjun; Oladejo, Babayemi O; Chai, Yan; Bi, Yuhai; Lu, Shan; Dong, Mengqiu; Zhang, Chang; Huang, Guangmei; Wong, Gary; Li, Na; Zhang, Yanfang; Li, Yan; Feng, Wen-Hai; Shi, Yi; Liang, Mifang; Zhang, Rongguang; Qi, Jianxun; Gao, George F

    2017-09-05

    Severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV) are two arthropod-borne phleboviruses in the Bunyaviridae family, which cause severe illness in humans and animals. Glycoprotein N (Gn) is one of the envelope proteins on the virus surface and is a major antigenic component. Despite its importance for virus entry and fusion, the molecular features of the phleboviruse Gn were unknown. Here, we present the crystal structures of the Gn head domain from both SFTSV and RVFV, which display a similar compact triangular shape overall, while the three subdomains (domains I, II, and III) making up the Gn head display different arrangements. Ten cysteines in the Gn stem region are conserved among phleboviruses, four of which are responsible for Gn dimerization, as revealed in this study, and they are highly conserved for all members in Bunyaviridae Therefore, we propose an anchoring mode on the viral surface. The complex structure of the SFTSV Gn head and human neutralizing antibody MAb 4-5 reveals that helices α6 in subdomain III is the key component for neutralization. Importantly, the structure indicates that domain III is an ideal region recognized by specific neutralizing antibodies, while domain II is probably recognized by broadly neutralizing antibodies. Collectively, Gn is a desirable vaccine target, and our data provide a molecular basis for the rational design of vaccines against the diseases caused by phleboviruses and a model for bunyavirus Gn embedding on the viral surface.

  8. Presynaptic neurones may contribute a unique glycoprotein to the extracellular matrix at the synapse

    Science.gov (United States)

    Caroni, Pico; Carlson, Steven S.; Schweitzer, Erik; Kelly, Regis B.

    1985-04-01

    As the extracellular matrix at the original site of a neuromuscular junction seems to play a major part in the specificity of synaptic regeneration1-5, considerable attention has been paid to unique molecules localized to this region6-11. Here we describe an extracellular matrix glycoprotein of the elasmobranch electric organ that is localized near the nerve endings. By immunological criteria, it is synthesized in the cell bodies, transported down the axons and is related to a glycoprotein in the synaptic vesicles of the neurones that innervate the electric organ. It is apparently specific for these neurones, as it cannot be detected elsewhere in the nervous system of the fish. Therefore, neurones seem to contribute unique extracellular matrix glycoproteins to the synaptic region. Synaptic vesicles could be involved in transporting these glycoproteins to or from the nerve terminal surface.

  9. Association study of the platelet collagen receptor glycoprotein VI gene with rheumatoid arthritis

    NARCIS (Netherlands)

    Michou, L.; Cornelis, F.; Baron, M.; Bombardieri, S.; Balsa, A.; Westhovens, R.; Barrera, P.; Alves, H.; Radstake, T.R.D.J.; Migliorini, P.; Bardin, T.; Petit-Teixeira, E.; Boilard, E.

    2013-01-01

    OBJECTIVES: Beyond their role in haemostasis, platelets can actively contribute to immunity. The activation of the platelet collagen receptor glycoprotein VI (GPVI) promotes the release of small extracellular vesicles called microparticles. These microparticles are found in the joint bathing fluid

  10. A Method for Determining the Content of Glycoproteins in Biological Samples

    Directory of Open Access Journals (Sweden)

    Yang Gao

    2016-11-01

    Full Text Available The glycoprotein purified from the mycelium extract of Tremella fuciformis was marked with iodine through the iodine substitution reaction. The content of iodine, which is indicative of the amount of the marked tremella glycoprotein (ITG, was detected with Inductively coupled plasma mass spectrometry (ICP-MS. The method was found to be stable, sensitive, and accurate at detecting the content of iodine-substituted glycoprotein, and was used in the quantitative analysis of biological samples, including blood and organs. Different biological samples were collected from rats after oral administration of ITG, and were tested for iodine content by ICP-MS to calculate the amount of ITG in the samples. The results suggested that ICP-MS is a sensitive, stable, and accurate method for detection of iodinated glycoproteins in blood and organs.

  11. Glycoproteins functionalized natural and synthetic polymers for prospective biomedical applications: A review.

    Science.gov (United States)

    Tabasum, Shazia; Noreen, Aqdas; Kanwal, Arooj; Zuber, Mohammad; Anjum, Muhammad Naveed; Zia, Khalid Mahmood

    2017-05-01

    Glycoproteins have multidimensional properties such as biodegradability, biocompatibility, non-toxicity, antimicrobial and adsorption properties; therefore, they have wide range of applications. They are blended with different polymers such as chitosan, carboxymethyl cellulose (CMC), polyvinyl pyrrolidone (PVP), polycaprolactone (PCL), heparin, polystyrene fluorescent nanoparticles (PS-NPs) and carboxyl pullulan (PC) to improve their properties like thermal stability, mechanical properties, resistance to pH, chemical stability and toughness. Considering the versatile charateristics of glycoprotein based polymers, this review sheds light on synthesis and characterization of blends and composites of glycoproteins, with natural and synthetic polymers and their potential applications in biomedical field such as drug delivery system, insulin delivery, antimicrobial wound dressing uses, targeting of cancer cells, development of anticancer vaccines, development of new biopolymers, glycoproteome research, food product and detection of dengue glycoproteins. All the technical scientific issues have been addressed; highlighting the recent advancement. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Immunoinformatic Analysis of Crimean Congo Hemorrhagic Fever Virus Glycoproteins and Epitope Prediction for Synthetic Peptide Vaccine.

    Science.gov (United States)

    Tipu, Hamid Nawaz

    2016-02-01

    To determine the Crimean Congo Hemorrhagic Fever (CCHF) virus M segement glycoprotein's immunoinformatic parameters, and identify Human Leukocyte Antigen (HLA) class I binders as candidates for synthetic peptide vaccines. Cross-sectional study. Combined Military Hospital, Khuzdar Cantt, in May 2015. Data acquisition, antigenicity prediction, secondary and tertiary structure prediction, residue analysis were done using immunoinformatics tools. HLAclass I binders in glycoprotein's sequence were identified at nanomer length using NetMHC 3.4 and mapped onto tertiary structure. Docking was done for strongest binder against its corresponding allele with CABS-dock. HLAA*0101, 0201, 0301, 2402, 2601 and B*0702, 0801, 2705, 3901, 4001, 5801, 1501 were analyzed against two glycoprotein components of the virus. Atotal of 35 nanomers from GP1, and 3 from GP2 were identified. HLAB*0702 bound maximum number of peptides (6), while HLAB*4001 showed strongest binding affinity. HLAspecific glycoproteins epitope prediction can help identify synthetic peptide vaccine candidates.

  13. Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

    DEFF Research Database (Denmark)

    Giang, Erick; Dorner, Marcus; Prentoe, Jannick C

    2012-01-01

    Hepatitis C virus (HCV) infects ∼2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and a...

  14. Comparative Studies of Vertebrate Platelet Glycoprotein 4 (CD36

    Directory of Open Access Journals (Sweden)

    Roger S. Holmes

    2012-09-01

    Full Text Available Platelet glycoprotein 4 (CD36 (or fatty acyl translocase [FAT], or scavenger receptor class B, member 3 [SCARB3] is an essential cell surface and skeletal muscle outer mitochondrial membrane glycoprotein involved in multiple functions in the body. CD36 serves as a ligand receptor of thrombospondin, long chain fatty acids, oxidized low density lipoproteins (LDLs and malaria-infected erythrocytes. CD36 also influences various diseases, including angiogenesis, thrombosis, atherosclerosis, malaria, diabetes, steatosis, dementia and obesity. Genetic deficiency of this protein results in significant changes in fatty acid and oxidized lipid uptake. Comparative CD36 amino acid sequences and structures and CD36 gene locations were examined using data from several vertebrate genome projects. Vertebrate CD36 sequences shared 53–100% identity as compared with 29–32% sequence identities with other CD36-like superfamily members, SCARB1 and SCARB2. At least eight vertebrate CD36 N-glycosylation sites were conserved which are required for membrane integration. Sequence alignments, key amino acid residues and predicted secondary structures were also studied. Three CD36 domains were identified including cytoplasmic, transmembrane and exoplasmic sequences. Conserved sequences included N- and C-terminal transmembrane glycines; and exoplasmic cysteine disulphide residues; TSP-1 and PE binding sites, Thr92 and His242, respectively; 17 conserved proline and 14 glycine residues, which may participate in forming CD36 ‘short loops’; and basic amino acid residues, and may contribute to fatty acid and thrombospondin binding. Vertebrate CD36 genes usually contained 12 coding exons. The human CD36 gene contained transcription factor binding sites (including PPARG and PPARA contributing to a high gene expression level (6.6 times average. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate CD36 gene with vertebrate

  15. Defining the Antigenic Structure of the Henipavirus Attachment (G) Glycoprotein: Implications for the Fusion Mechanism

    Science.gov (United States)

    2009-01-01

    ENSCO) event limited available foodstuffs and roosting habitat for the bats (30). Ultimately, the bats began foraging for food at orchards often...the medium was replaced once prior to screening colony supernatant by ELISA with sG glycoprotein antigen. To ensure clonal cultures, colonies were...immunized with sGNiV glycoprotein. Identification of neutralizing mAbs To identify fusion inhibitory mAbs, supernatant harvested from each clonal

  16. The quality control of glycoprotein folding in the endoplasmic reticulum, a trip from trypanosomes to mammals

    Directory of Open Access Journals (Sweden)

    A.J. Parodi

    1998-05-01

    Full Text Available The present review deals with the stages of synthesis and processing of asparagine-linked oligosaccharides occurring in the lumen of the endoplasmic reticulum and their relationship to the acquisition by glycoproteins of their proper tertiary structures. Special emphasis is placed on reactions taking place in trypanosomatid protozoa since their study has allowed the detection of the transient glucosylation of glycoproteins catalyzed by UDP-Glc:glycoprotein glucosyltransferase and glucosidase II. The former enzyme has the unique property of covalently tagging improperly folded conformations by catalyzing the formation of protein-linked Glc1Man7GlcNAc2, Glc1Man8GlcNac2 and Glc1Man9GlcNAc2 from the unglucosylated proteins. Glucosyltransferase is a soluble protein of the endoplasmic reticulum that recognizes protein domains exposed in denatured but not in native conformations (probably hydrophobic amino acids and the innermost N-acetylglucosamine unit that is hidden from macromolecular probes in most native glycoproteins. In vivo, the glucose units are removed by glucosidase II. The influence of oligosaccharides in glycoprotein folding is reviewed as well as the participation of endoplasmic reticulum chaperones (calnexin and calreticulin that recognize monoglucosylated species in the same process. A model for the quality control of glycoprotein folding in the endoplasmic reticulum, i.e., the mechanism by which cells recognize the tertiary structure of glycoproteins and only allow transit to the Golgi apparatus of properly folded species, is discussed. The main elements of this control are calnexin and calreticulin as retaining components, the UDP-Glc:glycoprotein glucosyltransferase as a sensor of tertiary structures and glucosidase II as the releasing agent.

  17. Rabies virus (RV) glycoprotein expression levels are not critical for pathogenicity of RV.

    Science.gov (United States)

    Wirblich, Christoph; Schnell, Matthias J

    2011-01-01

    Previous comparisons of different rabies virus (RV) strains suggested an inverse relationship between pathogenicity and the amount of glycoprotein produced in infected cells. In order to provide more insight into this relationship, we pursued an experimental approach that allowed us to alter the glycoprotein expression level without altering the glycoprotein sequence, thereby eliminating the contribution of amino acid changes to differences in viral virulence. To this end, we constructed an infectious clone of the highly pathogenic rabies virus strain CVS-N2c and replaced its cognate glycoprotein gene with synthetic versions in which silent mutations were introduced to replace wild-type codons with the most or least frequently used synonymous codons. A recombinant N2c variant containing the fully codon-optimized G gene and three variants carrying a partially codon-deoptimized G gene were recovered on mouse neuroblastoma cells and shown to express 2- to 3-fold more and less glycoprotein, respectively, than wild-type N2c. Pathogenicity studies in mice revealed the WT-N2c virus to be the most pathogenic strain. Variants containing partially codon-deoptimized glycoprotein genes or the codon-optimized gene were less pathogenic than WT-N2c but still caused significant mortality. We conclude that the expression level of the glycoprotein gene does have an impact on pathogenicity but is not a dominant factor that determines pathogenicity. Thus, strategies such as changes in codon usage that aim solely at altering the expression level of the glycoprotein gene do not suffice to render a pathogenic rabies virus apathogenic and are not a viable and safe approach for attenuation of a pathogenic strain.

  18. Characterization and cloning of fasciclin I and fasciclin II glycoproteins in the grasshopper

    OpenAIRE

    Snow, Peter M.; Zinn, Kai; Harrelson, Allan L.; McAllister, Linda; Schilling, Jim; Bastiani, Michael J.; Makk, George; Goodman, Corey S.

    1988-01-01

    Monoclonal antibodies were previously used to identify two glycoproteins, called fasciclin I and II (70 and 95 kDa, respectively), which are expressed on different subsets of axon fascicles in the grasshopper (Schistocerca americana) embryo. Here the monoclonal antibodies were used to purify these two membrane-associated glycoproteins for further characterization. Fasciclin II appears to be an integral membrane protein, whereas fasciclin I is an extrinsic membrane protein. The amino acid sequ...

  19. Isolation and partial chemical characterization of a 64,000-dalton glycoprotein of human cytomegalovirus

    International Nuclear Information System (INIS)

    Clark, B.R.; Zaia, J.A.; Balce-Directo, L.; Ting, Y.P.

    1984-01-01

    A guanidinium chloride extract of [ 3 H]glucosamine- and [ 35 S]methionine-labeled virions plus dense bodies of human cytomegalovirus (Towne) was separated by reverse-phase high-pressure liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the eluate revealed the major peak to be a glycoprotein with a relative mass of 64,000. This glycoprotein (HCMVgp64) was characterized by amino acid analysis and a high-pressure liquid chromatographic map of its tryptic peptides

  20. Characterisation and evaluation of antiviral recombinant peptides based on the heptad repeat regions of NDV and IBV fusion glycoproteins

    International Nuclear Information System (INIS)

    Wang Xiaojia; Li Chuangen; Chi Xiaojing; Wang Ming

    2011-01-01

    Mixed virus infections can cause livestock losses that are more devastating than those caused by single virus infections. Newcastle disease virus (NDV) and infectious bronchitis virus (IBV), serious threats to the poultry industry, can give rise to complex mixed infections that hinder diagnosis and prevention. In this study, we show that newly designed peptides, which are based on the heptad repeat (HR) region of the fusion glycoproteins from NDV and IBV, have more potent antiviral activity than the mother HR peptides. Plaque formation and chicken embryo infectivity assays confirmed these results. The novel peptides completely inhibited single virus infections and mixed infections caused by NDV and IBV. Furthermore, we assessed cell toxicity and possible targets for the peptides, thereby strengthening the notion that HR2 is an attractive site for therapeutic intervention. These results suggest the possibility of designing a relatively broad-spectrum class of antiviral peptides that can reduce the effects of mixed-infections.

  1. Identification of a new mutation in platelet glycoprotein IX (GPIX) in a patient with Bernard-Soulier syndrome.

    Science.gov (United States)

    Rivera, C E; Villagra, J; Riordan, M; Williams, S; Lindstrom, K J; Rick, M E

    2001-01-01

    We describe a new mutation in glycoprotein IX (GPIX) in a patient with Bernard-Soulier syndrome (BSS). Sequencing of GPIX revealed a homozygous (T-->C) transition at nucleotide 1717 (GenBank/HUMGPIX/M80478), resulting in a Cys(8) (TGT)-->Arg (CGT) replacement in the mature peptide. DNA restriction enzyme analysis using BsaAI revealed that the patient was homozygous and that his parents were heterozygous for the defect. This mutation disrupts a putative disulphide bond between the Cys(8) and Cys(12) that would alter the secondary structure of GPIX and which probably accounts for the absence of the GPIb/IX/V complex from the platelet surface in this patient.

  2. [Thrombotic microangiopathies: HUS/TTP. Physiopathological aspects].

    Science.gov (United States)

    Sánchez Avalos, J C

    2000-01-01

    In thrombotic thrombocytopenic purpura (TTP) and in the hemolytic uremic syndrome (HUS) fibrin-platelet thrombi occlude arterioles and capillaries. The mechanism of endothelial cell injury and the mechanism of thrombosis are the most important physiopathological events in this pathology and are largely unknown. In HUS due to the Shiga toxin, the lesion of the endothelial cells is due to penetration of the toxin into the cell via the Gb3 receptor. Endothelial cell death is a consequence of altered protein synthesis at the ribosomal level. Cytokines released during the inflammatory process, possibly enhance the endothelial damage. Genetic and immunologic predisposing factors for the development of HUS have also been postulated. In idiopathic, secondary and familial HUS/TTP the mechanism of endothelial lesion is unknown, but multiple responsible factors have been advocated such as infections, drugs, pregnancy, autoantibodies, apoptosis inducing molecules, etc. and other genetic, hormonal or immunologic predisposing factors may also be involved. Factor H deficiency has been blamed in familiar cases. The most important cause of microcirculation thrombosis is the thrombogenic capacity of endothelial cell "activation" or injury induced by multiple mechanisms. The predominant source of plasma vW factor multimers is apparent in the altered endothelial cell. The unusually large vWF multimers are more effective at binding to platelet glycoprotein Ib-IX and IIb-IIIa complexes and inducing aggregation, as also occurred with the low weight multimers formed with excessive proteolysis, as described in the acute phase of HUS/TTP. The recent report of congenital deficiency of a vWF protease in familial TTP and its functional inhibition by autoantibodies in acquired cases is characteristic of TTP. This protease inhibition has never been described in HUS and might represent pathogenetic differences between TTP and HUS, and contribute to the differential diagnosis, but further

  3. The sweet spot for biologics: recent advances in characterization of biotherapeutic glycoproteins.

    Science.gov (United States)

    O'Flaherty, Róisín; Trbojević-Akmačić, Irena; Greville, Gordon; Rudd, Pauline M; Lauc, Gordan

    2018-01-01

    Glycosylation is recognized as a Critical Quality Attribute for therapeutic glycoproteins such as monoclonal antibodies, fusion proteins and therapeutic replacement enzymes. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for their discovery, development and quality control. The aim of this review is to highlight relevant and recent advances in analytical technologies for characterization of biotherapeutic glycoproteins. Areas covered: The review gives an overview of the glycosylation trends of biotherapeutics approved in 2016 and 2017 by FDA. It describes current and novel analytical technologies for characterization of therapeutic glycoproteins and is explored in the context of released glycan, glycopeptide or intact glycoprotein analysis. Ultra performance liquid chromatography, mass spectrometry and capillary electrophoresis technologies are explored in this context. Expert commentary: There is a need for the biopharmaceutical industry to incorporate novel state of the art analytical technologies into existing and new therapeutic glycoprotein workflows for safer and more efficient biotherapeutics and for the improvement of future biotherapeutic design. Additionally, at present, there is no 'gold-standard' approach to address all the regulatory requirements and as such this will involve the use of orthogonal glycoanalytical technologies with a view to gain diagnostic information about the therapeutic glycoprotein.

  4. The nature of interactions between tissue-type plasminogen activator and platelets

    International Nuclear Information System (INIS)

    Torr, S.R.; Winters, K.J.; Santoro, S.A.; Sobel, B.E.

    1990-01-01

    To elucidate interactions responsible for inhibition of aggregation of platelets in platelet-rich plasma (PRP) harvested from whole blood preincubated with t-PA, experiments were performed with PRP and washed platelets under diverse conditions of preincubation. Both ADP and collagen induced aggregation were inhibited in PRP unless aprotinin had been added to the preincubated whole blood concomitantly with t-PA. However, in washed platelets prepared after the same exposure aggregation was intact. When washed platelets were supplemented with fibrinogen degradation products (FDPs) in concentrations simulating those in whole blood preincubated with t-PA, aggregation induced with either ADP or collagen was inhibited. Thus, the inhibition in PRP depended on generation of FDPs by activated plasminogen. The functional integrity of surface glycoprotein (GP) IIb/IIIa receptors in washed platelets was documented by autoradiography after SDS-PAGE of surface labeled GPs and by fibrinogen binding despite preincubation of the whole blood or washed platelets themselves with t-PA and plasminogen as long as exogenous calcium (greater than or equal to 0.1 microM) was present. In contrast, when calcium was absent, the platelet GP IIb/IIIa receptor was rendered susceptible to degradation by plasmin, and aggregation was inhibited by preincubation at 37 degrees C even if aprotinin was present when aggregation was being assayed. These observations reconcile disparate results in the literature from studies in vivo and in vitro by demonstrating that inhibition of aggregation of platelets in PRP and in whole blood reflects indirect effects of plasminogen activation rather than direct effects of t-PA or plasmin on the platelets themselves

  5. Bivalirudin in percutaneous coronary intervention

    Directory of Open Access Journals (Sweden)

    Sam J Lehman

    2006-12-01

    Full Text Available Sam J Lehman, Derek P ChewDepartment of Medicine, Flinders University, South Australia, AustraliaAbstract: Bivalirudin is a member of the direct thrombin inhibitor group of anticoagulants. It has been evaluated as an alternative to unfractionated and low-molecular-weight heparins in the settings of percutaneous coronary intervention (PCI and acute coronary syndrome (ACS. Results of clinical trials to date suggest bivalirudin is a viable alternative to the use of a heparin combined with a glycoprotein (GP IIb/IIIa inhibitor in these settings. Thrombin has a central role in coagulation and platelet activation in ACS and during PCI. Its direct inhibition is an attractive target for therapy in these settings. Bivalirudin is a 20 amino acid polypeptide hirudin analog. It displays bivalent and reversible binding to the thrombin molecule, inhibiting its action. Direct inhibition of thrombin with bivalirudin has theoretical pharmacokinetic and pharmacodynamic advantages over the indirect anticoagulants. A reduction in rates of bleeding without loss of anti-thrombotic efficacy has been a consistent finding across multiple clinical trials. There may be economic benefits to the use of bivalirudin if it permits a lower rate of use of the GP IIb/IIIa inhibitors. This article reviews the pharmacology of bivalirudin and clinical trial evidence to date. There are now data from multiple clinical trials and meta-analyses in the setting of ACS and PCI. Early results from the acute catheterization and urgent intervention strategy (ACUITY trial are discussed. Keywords: bivalirudin, direct thrombin inhibitor, acute coronary syndrome, percutaneous coronary intervention

  6. Thrombolytic Effects of the Snake Venom Disintegrin Saxatilin Determined by Novel Assessment Methods: A FeCl3-Induced Thrombosis Model in Mice

    Science.gov (United States)

    Kim, Young Dae; Nam, Hyo Suk; Kang, Sungsoo; Yang, Seung-Hee; Heo, Ji Hoe

    2013-01-01

    Saxatilin, a novel disintegrin purified and cloned from the venom of the Korean snake Gloydius saxatilis, strongly inhibits activation and aggregation of platelets. Glycoprotein (GP) IIb/IIIa receptor antagonists can resolve thrombus, so saxatilin might also have thrombolytic effects. We investigated the thrombolytic effects of saxatilin in mice using a ferric chloride-induced carotid arterial thrombosis model. Thrombotic occlusion and thrombus resolution were evaluated quantitatively by measuring blood flow in the carotid artery with an ultrasonic flow meter and calculating the degree of flow restoration on a minute-by-minute basis; results were confirmed by histological examination. Saxatilin dissolved thrombi in a dose-dependent manner. Saxatilin at 5 mg/kg restored blood flow to baseline levels. As saxatilin dose increased, time to recanalization decreased. A bolus injection of 10% of a complete dose with continuous infusion of the remaining dose for 60 minutes resulted in effective recanalization without reocclusion. The thrombolytic effect of saxatilin was also demonstrated in vitro using platelet aggregometry by administering saxatilin in preformed thrombi. Bleeding complications were observed in 2 of 71 mice that received saxatilin. Fibrin/fibrinogen zymography and platelet aggregometry studies indicated that saxatilin does not have fibrinolytic activity, but exerted its action on platelets. Integrin-binding assays showed that saxatilin inhibited multiple integrins, specifically α2bβ3 (GP IIb/IIIa), α5β1, αvβ3, αvβ1, and αvβ5, which act on platelet adhesion/aggregation. Saxatilin inhibited multiple integrins by acting on platelets, and was safe and effective in resolving thrombi in mice. PMID:24260554

  7. Four glycoproteins are expressed in the cat zona pellucida.

    Science.gov (United States)

    Stetson, I; Avilés, M; Moros, C; García-Vázquez, F A; Gimeno, L; Torrecillas, A; Aliaga, C; Bernardo-Pisa, M V; Ballesta, J; Izquierdo-Rico, M J

    2015-04-15

    The mammalian oocyte is surrounded by a matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding and may be involved in speciation. In cat (Felis catus), this matrix is composed of at least three glycoproteins called ZP2, ZP3, and ZP4. However, recent studies have pointed to the presence of a fourth protein in several mammals (rat, human, hamster or rabbit), meaning that a reevaluation of cat ZP is needed. For this reason, the objective of this research was to analyze the protein composition of cat ZP by means of proteomic analysis. Using ZP from ovaries and oocytes, several peptides corresponding to four proteins were detected, yielding a coverage of 33.17%, 71.50%, 50.23%, and 49.64% for ZP1, ZP2, ZP3, and ZP4, respectively. Moreover, the expression of four genes was confirmed by molecular analysis. Using total RNA isolated from cat ovaries, the complementary deoxyribonucleic acids encoding cat ZP were partially amplified by reverse-transcribed polymerase chain reaction. Furthermore, ZP1 was totally amplified for the first time in this species. As far as we are aware, this is the first study that confirms the presence of four proteins in cat ZP. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Antifreeze Glycoproteins Alter the Molecular Scale Surface Morphology of Ice

    Science.gov (United States)

    Zepeda, Salvador; Orme, Christine A.; Qiu, Roger; Yeh, Yin

    2003-03-01

    Trematomas borchgrevinki live in the harsh super-cooled waters of the Antarctic. Critical to their survival are antifreeze glycoproteins (AFGPs) that further suppress the freezing temperature of their blood serum in addition to the colligative action of salts found in the ocean. These proteins also modify ice crystal growth habits as well as inhibit recrystallization in polycrystalline ice. To date many other types of antifreeze proteins have been identified in cold weather insects, plants, and other fish, but the exact mechanism is not entirely understood. The mechanism is non-colligative since only a few mg/ml are required for ice crystal growth inhibition and a non-equilibrium melting/freezing point hysteresis is observed. Atomic force microscopy (AFM) can yield a wealth of surface information that can reveal molecular scale information of biomineralization processes. We use AFM to directly probe the surface of ice crystals grown from the vapor in the pure phase and in the presence of growth inhibitors/modifiers, AFGPs. Results show that the AFGPs heavily pin the surface of ice.

  9. Glycoproteins and Glycosylation Site Assignments in Cereal seed Proteomes

    DEFF Research Database (Denmark)

    Dedvisitsakul, Plaipol

    The study of plant proteomes is important to further the understanding of biological processes and enhance the agronomical and nutritional value of crops and food products. To gain deeper understanding on the proteome level, it is important to characterize posttranslational modifications. Glycosy......The study of plant proteomes is important to further the understanding of biological processes and enhance the agronomical and nutritional value of crops and food products. To gain deeper understanding on the proteome level, it is important to characterize posttranslational modifications...... by supplementing cotton wool with ZIC-HILIC in a microcolumn (called ZIC-cotton). This approach reduced co-enrichment of non-glycosylated peptides and allowed glycoppeptide identification from large protein mixtures. It was applied for glycoprotein identification and glycosylation site assignment in wheat albumin...... and barley aleurone layer proteins. By sitespecific glycosylation labeling and LC-MS/MS analysis, 76 different glycosylation sites within 65 wheat albumin proteins were identified using a combination of ZIC-cotton and cotton wool. In addition, ZIC-cotton has been also applied to proteins produced from barley...

  10. Rabies virus glycoprotein as a carrier for anthrax protective antigen

    International Nuclear Information System (INIS)

    Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen was also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems

  11. Urine Glycoprotein Profile Reveals Novel Markers for Chronic Kidney Disease

    Directory of Open Access Journals (Sweden)

    Anuradha Vivekanandan-Giri

    2011-01-01

    Full Text Available Chronic kidney disease (CKD is a significant public health problem, and progression to end-stage renal disease leads to dramatic increases in morbidity and mortality. The mechanisms underlying progression of disease are poorly defined, and current noninvasive markers incompletely correlate with disease progression. Therefore, there is a great need for discovering novel markers for CKD. We utilized a glycoproteomic profiling approach to test the hypothesis that the urinary glycoproteome profile from subjects with CKD would be distinct from healthy controls. N-linked glycoproteins were isolated and enriched from the urine of healthy controls and subjects with CKD. This strategy identified several differentially expressed proteins in CKD, including a diverse array of proteins with endopeptidase inhibitor activity, protein binding functions, and acute-phase/immune-stress response activity supporting the proposal that inflammation may play a central role in CKD. Additionally, several of these proteins have been previously linked to kidney disease implicating a mechanistic role in disease pathogenesis. Collectively, our observations suggest that the human urinary glycoproteome may serve as a discovery source for novel mechanism-based biomarkers of CKD.

  12. Characterization of sialylated and fucosylated glycopeptides of beta2-glycoprotein I by a combination of HILIC LC and MALDI MS/MS

    DEFF Research Database (Denmark)

    Kondo, Akira; Thaysen-Andersen, Morten; Hjernø, Karin

    2010-01-01

    features of RP (Poros R2) and hydrophilic (zwitter-ionic hydrophilic interaction chromatography) chromatographic resins. The glycopeptide analysis workflow was applied to human beta2-glycoprotein I (beta2-GPI, apolipoprotein H), which contains multiple N-glycosylation sites. Conditions for rapid...... were characterized using MALDI quadrupole TOF MS/MS. A total of 23 glycan structures, including sialylated bi- and tri-antennary complex type glycans, were characterized at three N-glycosylation sites, namely Asn-143, Asn-174 and Asn-234, of beta2-GPI. Further exploration of the complementary nature...

  13. Recombinant N-Domain of Pregnancy-Specific Glycoprotein from E. coli Cells: Analysis of the Spectrum of Polyclonal Antibodies.

    Science.gov (United States)

    Prokopenko, P G; Shkoporov, A N; Petrenko, O Yu; Efimov, B A; Negrebetskii, V V; Terent'ev, A A

    2015-11-01

    We studied antibody spectrum in antisera to IgG-like recombinant N-domain of pregnancyspecific glycoprotein-1 (rPSG-N) from E. coli cells. In three experimental series, the fraction of IgG antibodies from anti-rPSG-N sera was immobilized on 3 immunoadsorbents: by polymerization with glutaraldehyde, on glutaraldehyde activated biogel P-300, and on commercial CNBr-activated 4B sepharose. Retroplacental serum was incubated with immobilized antibodies to rPSG1-N, protein was eluted and tested in the precipitation test in standard test systems with PSG1, IgG, and human serum albumin. Three proteins were eluted from all 3 immunoadsorbents: PSG1, IgG, and human serum albumin, which demonstrated the spectrum of antibodies to 3 proteins present also in natural serum PSG1 complex. The proportions of PSG1 and IgG obtained in these experiments were similar to those in natural serum PSG1 complex, while the level of human serum albumin was significantly higher in natural PSG1 complex. Thus, we failed to obtain PSG1 monoprotein free from IgG and human serum albumin. Antigenic mosaicism of the polypeptide chain of IgG-like rPSG1-N relative to the antigenic polyvalence of the complex of three proteins present in bioactive preparation of natural serum PSG1 was discussed.

  14. Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH.

    Science.gov (United States)

    Vallbracht, Melina; Rehwaldt, Sascha; Klupp, Barbara G; Mettenleiter, Thomas C; Fuchs, Walter

    2018-05-01

    Many viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reduced in vitro fusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in the Varicellovirus genus, resulted in enhanced in vitro fusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. IMPORTANCE Herpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide

  15. Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

    Science.gov (United States)

    Kim, In-Joong; Chouljenko, Vladimir N.; Walker, Jason D.

    2013-01-01

    Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread. PMID:23678175

  16. 3,3′,4,4′,5-Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein

    Directory of Open Access Journals (Sweden)

    Hiroshi Fujise

    2004-01-01

    Full Text Available The effects on the drug efflux of 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126, the most toxic of all coplanar polychlorinated biphenyls (Co-PCBs, were examined in KB-3 cells expressing human wild-type and mutant P-glycoprotein in which the 61st amino acid was substituted for serine or phenylalanine (KB3-Phe61. In the cells expressing P-glycoproteins, accumulations of vinblastine and colchicine decreased form 85% to 92% and from 62% to 91%, respectively, and the drug tolerances for these chemicals were increased. In KB3-Phe61, the decreases in drug accumulation were inhibited by adding PCB-126 in a way similar to that with cyclosporine A: by adding 1 μM PCB-126, the accumulations of vinblastine and colchicine increased up to 3.3- and 2.3-fold, respectively. It is suggested that PCB-126 decreased the drug efflux by inhibiting the P-glycoprotein in KB3-Phe61. Since there were various P-glycoproteins and many congeners of Co-PCBs, this inhibition has to be considered a new cause of the toxic effects of Co-PCBs.

  17. The Role of the Hendra Virus and Nipah Virus Attachment Glycoproteins in Receptor Binding and Antibody Neutralization

    Science.gov (United States)

    2014-01-31

    glycoproteins, such as HIV gp140 and Rabies glycoprotein, its ability to form trimers but not tetramers makes it ineffective for correct oligomerization of...Oldstone MB. 2000. V and C proteins of measles virus function as virulence factors in vivo. Virology 267:80-9.   216 148. Playford EG, McCall B...2005. Stable trimerization of recombinant rabies virus glycoprotein ectodomain is required for interaction with the p75NTR receptor. J Gen Virol 86

  18. Overview of P-glycoprotein inhibitors: a rational outlook

    Directory of Open Access Journals (Sweden)

    Kale Mohana Raghava Srivalli

    2012-09-01

    Full Text Available P-glycoprotein (P-gp, a transmembrane permeability glycoprotein, is a member of ATP binding cassette (ABC super family that functions specifically as a carrier mediated primary active efflux transporter. It is widely distributed throughout the body and has a diverse range of substrates. Several vital therapeutic agents are substrates to P-gp and their bioavailability is lowered or a resistance is induced because of the protein efflux. Hence P-gp inhibitors were explored for overcoming multidrug resistance and poor bioavailability problems of the therapeutic P-gp substrates. The sensitivity of drug moieties to P-gp and vice versa can be established by various experimental models in silico, in vitro and in vivo. Ever since the discovery of P-gp, the research plethora identified several chemical structures as P-gp inhibitors. The aim of this review was to emphasize on the discovery and development of newer, inert, non-toxic, and more efficient, specifically targeting P-gp inhibitors, like those among the natural herb extracts, pharmaceutical excipients and formulations, and other rational drug moieties. The applications of cellular and molecular biology knowledge, in silico designed structural databases, molecular modeling studies and quantitative structure-activity relationship (QSAR analyses in the development of novel rational P-gp inhibitors have also been mentioned.Glicoproteína-p (P-gp, uma glicoproteína de transmembrana permeável, é um membro da superfamília (ABC de cassete de gene de ligação de ATP que funciona especificamente como um carreador mediado pelo transportador de efluxo ativo primário. É amplamente distribuído por todo o corpo e apresenta uma gama diversificada de substratos. Diversos agentes terapêuticos vitais são substratos para P-gp e sua biodisponibilidade é reduzida ou a resistência é induzida devido ao efluxo de proteínas. Portanto, os inibidores da P-gp foram explorados para a superação da resistência a

  19. A novel function of N-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection.

    Directory of Open Access Journals (Sweden)

    Takuya Kanno

    Full Text Available Therapeutic agents to the central nervous system (CNS need to be efficiently delivered to the target site of action at appropriate therapeutic levels. However, a limited number of effective drugs for the treatment of neurological diseases has been developed thus far. Further, the pharmacological mechanisms by which such therapeutic agents can protect neurons from cell death have not been fully understood. We have previously reported the novel small-molecule compound, 2-[mesityl(methylamino]-N-[4-(pyridin-2-yl-1H-imidazol-2-yl] acetamide trihydrochloride (WN1316, as a unique neuroprotectant against oxidative injury and a highly promising remedy for the treatment of amyotrophic lateral sclerosis (ALS. One of the remarkable characteristics of WN1316 is that its efficacious doses in ALS mouse models are much less than those against oxidative injury in cultured human neuronal cells. It is also noted that the WN1316 cytoprotective activity observed in cultured cells is totally dependent upon the addition of fetal bovine serum in culture medium. These findings led us to postulate some serum factors being tightly linked to the WN1316 efficacy. In this study, we sieved through fetal bovine serum proteins and identified two N-linked glycoproteins, alpha-2-HS-glycoprotein (AHSG and hemopexin (HPX, requisites to exert the WN1316 cytoprotective activity against oxidative injury in neuronal cells in vitro. Notably, the removal of glycan chains from these molecules did not affect the WN1316 cytoprotective activity. Thus, two glycoproteins, AHSG and HPX, represent a pivotal glycoprotein of the cytoprotective activity for WN1316, showing a concrete evidence for the novel glycan-independent function of serum glycoproteins in neuroprotective drug efficacy.

  20. Thyroid Hormone and P-Glycoprotein in Tumor Cells

    Directory of Open Access Journals (Sweden)

    Paul J. Davis

    2015-01-01

    Full Text Available P-glycoprotein (P-gp; multidrug resistance pump 1, MDR1; ABCB1 is a plasma membrane efflux pump that when activated in cancer cells exports chemotherapeutic agents. Transcription of the P-gp gene (MDR1 and activity of the P-gp protein are known to be affected by thyroid hormone. A cell surface receptor for thyroid hormone on integrin αvβ3 also binds tetraiodothyroacetic acid (tetrac, a derivative of L-thyroxine (T4 that blocks nongenomic actions of T4 and of 3,5,3′-triiodo-L-thyronine (T3 at αvβ3. Covalently bound to a nanoparticle, tetrac as nanotetrac acts at the integrin to increase intracellular residence time of chemotherapeutic agents such as doxorubicin and etoposide that are substrates of P-gp. This action chemosensitizes cancer cells. In this review, we examine possible molecular mechanisms for the inhibitory effect of nanotetrac on P-gp activity. Mechanisms for consideration include cancer cell acidification via action of tetrac/nanotetrac on the Na+/H+ exchanger (NHE1 and hormone analogue effects on calmodulin-dependent processes and on interactions of P-gp with epidermal growth factor (EGF and osteopontin (OPN, apparently via αvβ3. Intracellular acidification and decreased H+ efflux induced by tetrac/nanotetrac via NHE1 is the most attractive explanation for the actions on P-gp and consequent increase in cancer cell retention of chemotherapeutic agent-ligands of MDR1 protein.

  1. Myelin Oligodendrocyte Glycoprotein: Deciphering a Target in Inflammatory Demyelinating Diseases

    Directory of Open Access Journals (Sweden)

    Patrick Peschl

    2017-05-01

    Full Text Available Myelin oligodendrocyte glycoprotein (MOG, a member of the immunoglobulin (Ig superfamily, is a myelin protein solely expressed at the outermost surface of myelin sheaths and oligodendrocyte membranes. This makes MOG a potential target of cellular and humoral immune responses in inflammatory demyelinating diseases. Due to its late postnatal developmental expression, MOG is an important marker for oligodendrocyte maturation. Discovered about 30 years ago, it is one of the best-studied autoantigens for experimental autoimmune models for multiple sclerosis (MS. Human studies, however, have yielded controversial results on the role of MOG, especially MOG antibodies (Abs, as a biomarker in MS. But with improved detection methods using different expression systems to detect Abs in patients’ samples, this is meanwhile no longer the case. Using cell-based assays with recombinant full-length, conformationally intact MOG, several recent studies have revealed that MOG Abs can be found in a subset of predominantly pediatric patients with acute disseminated encephalomyelitis (ADEM, aquaporin-4 (AQP4 seronegative neuromyelitis optica spectrum disorders (NMOSD, monophasic or recurrent isolated optic neuritis (ON, or transverse myelitis, in atypical MS and in N-methyl-d-aspartate receptor-encephalitis with overlapping demyelinating syndromes. Whereas MOG Abs are only transiently observed in monophasic diseases such as ADEM and their decline is associated with a favorable outcome, they are persistent in multiphasic ADEM, NMOSD, recurrent ON, or myelitis. Due to distinct clinical features within these diseases it is controversially disputed to classify MOG Ab-positive cases as a new disease entity. Neuropathologically, the presence of MOG Abs is characterized by MS-typical demyelination and oligodendrocyte pathology associated with Abs and complement. However, it remains unclear whether MOG Abs are a mere inflammatory bystander effect or truly pathogenetic

  2. Molecular insight into conformational transmission of human P-glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Shan-Yan [Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Liu, Fu-Feng, E-mail: fufengliu@tju.edu.cn, E-mail: ysun@tju.edu.cn; Dong, Xiao-Yan; Sun, Yan, E-mail: fufengliu@tju.edu.cn, E-mail: ysun@tju.edu.cn [Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072 (China)

    2013-12-14

    P-glycoprotein (P-gp), a kind of ATP-binding cassette transporter, can export candidates through a channel at the two transmembrane domains (TMDs) across the cell membranes using the energy released from ATP hydrolysis at the two nucleotide-binding domains (NBDs). Considerable evidence has indicated that human P-gp undergoes large-scale conformational changes to export a wide variety of anti-cancer drugs out of the cancer cells. However, molecular mechanism of the conformational transmission of human P-gp from the NBDs to the TMDs is still unclear. Herein, targeted molecular dynamics simulations were performed to explore the atomic detail of the conformational transmission of human P-gp. It is confirmed that the conformational transition from the inward- to outward-facing is initiated by the movement of the NBDs. It is found that the two NBDs move both on the two directions (x and y). The movement on the x direction leads to the closure of the NBDs, while the movement on the y direction adjusts the conformations of the NBDs to form the correct ATP binding pockets. Six key segments (KSs) protruding from the TMDs to interact with the NBDs are identified. The relative movement of the KSs along the y axis driven by the NBDs can be transmitted through α-helices to the rest of the TMDs, rendering the TMDs to open towards periplasm in the outward-facing conformation. Twenty eight key residue pairs are identified to participate in the interaction network that contributes to the conformational transmission from the NBDs to the TMDs of human P-gp. In addition, 9 key residues in each NBD are also identified. The studies have thus provided clear insight into the conformational transmission from the NBDs to the TMDs in human P-gp.

  3. Convulxin binds to native, human glycoprotein Ib alpha.

    Science.gov (United States)

    Kanaji, Sachiko; Kanaji, Taisuke; Furihata, Kenichi; Kato, Kazunobu; Ware, Jerry L; Kunicki, Thomas J

    2003-10-10

    Convulxin (CVX), a C-type snake protein from Crotalus durissus terrificus venom, is the quintessential agonist for studies of the collagen receptor, glycoprotein VI (GPVI) and its role in platelet adhesion to collagens. In this study, CVX, purified from venom, behaves as expected, i.e. it binds to platelet GPVI and recombinant human GPVI, induces platelet aggregation and platelet prothrombinase activity, and binds uniquely to GPVI in ligand blots of SDS-denatured proteins. Nonetheless, we find that CVX has a dual specificity for both GPVI and native but not denatured human GPIb alpha. First, CVX binds to human GPIb alpha expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIb alpha but more strongly to human platelet GPIb alpha, as evidenced by comparative binding to wild-type, GPVI(-/-), FcR gamma (-/-), and human GPIb transgenic mice. Third, the binding of CVX to human GPIb alpha is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIb alpha is disrupted by phenylalanine substitutions at GPIb alpha tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and alpha-thrombin binding to GPIb alpha. Fifth, CVX binding to GPIb alpha on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIb alpha antibodies. Lastly, CVX fails to bind to denatured GPIb alpha in detergent extracts of platelets. Three separate preparations of CVX (two purified by the authors; one obtained commercially) produced equivalent results. These results indicate that CVX exhibits dual specificity for both native GPIb alpha and GPVI. Furthermore, the binding site on GPIb alpha for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIb alpha to CVX-induced platelet responses needs to be carefully re-evaluated.

  4. Multiple Drug Transport Pathways through human P-Glycoprotein(†)

    Science.gov (United States)

    McCormick, James W.; Vogel, Pia D.; Wise, John G.

    2015-01-01

    P-glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11 to 12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methyl-pyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar is presented that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp. PMID:26125482

  5. Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

    Directory of Open Access Journals (Sweden)

    Oliveira M.C.

    1999-01-01

    Full Text Available Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6% presented hypogonadism (which was central in 45 cases and primary in 2, 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01 and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02. Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01 as well as free testosterone levels (r = -0.53, P<0.01. In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

  6. Zinc alpha-2 glycoprotein is overproduced in Cushing's syndrome.

    Science.gov (United States)

    Escoté, Xavier; Aranda, Gloria B; Mora, Mireia; Casals, Gregori; Enseñat, Joaquim; Vidal, Oscar; Esteban, Yaiza; Halperin, Irene; Hanzu, Felicia A

    2017-01-01

    Cushing syndrome (CS), an endogenous hypercortisolemic condition with increased cardiometabolic morbidity, leads to development of abdominal obesity, insulin resistance, diabetes and proatherogenic dyslipidemia. Zinc alpha-2 glycoprotein (ZAG) is a recently characterized lipolytic adipokine implicated in regulation of adipose tissue metabolism and fat distribution. In vitro and animal studies suggest that glucocorticoids interact with ZAG secretion and action. To assess the relationship between ZAG and glucocorticoids in a human model of hypercortisolism, circulating ZAG levels were tested in patients with CS and its counterpart controls. An observational, cross-sectional study on 39 women, 13 with active CS and 26 controls matched by age and body mass index. Plasma ZAG levels (μg/ml) were measured by ELISA and correlated with hypercortisolism, metabolic, and phenotypic parameters. Plasma ZAG levels were significantly higher in patients with CS compared to controls (64.3±16.6 vs. 44.0±16.1, p=0.002). In a univariate analysis, ZAG levels positively correlated to 24-h urinary free cortisol (p=0.001), body mass index (p=0.02), non-esterified fatty acids (p=0.05), glucose (p=0.003), LDL-C (p=0.028), and type 2 diabetes mellitus (p=0.016), and were inversely related to total adiponectin levels (p=0.035). In a multivariate analysis, after adjusting for CS, ZAG levels only correlated with body mass index (p=0.012), type 2 diabetes mellitus (p=0.004), and glucose (p<0.001). This study provides initial evidence that plasma ZAG levels are higher in patients with CS as compared to controls. The close relationship of ZAG with metabolic and phenotypic changes in CS suggests that ZAG may play a significant role in adipose tissue changes in hypercortisolism. Copyright © 2017 SEEN. Publicado por Elsevier España, S.L.U. All rights reserved.

  7. Alterations of intestinal glycoprotein hydrolases in congenital diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Najjar, S.M.

    1989-01-01

    The diabetic BioBreed (BB{sub d}) rat was used for the study of the molecular structure of intestinal brush border sucrase-{alpha}-dextrinase (SD) and aminooligopeptidase (AOP) in diabetes mellitus. The specific catalytic activity of S-D and AOP in the BB{sub d} rat is normal. However, solid-phase radioimmunoassay revealed loss of some antigenic determinants in the BB{sub d} rat. S-D and AOP migrated abnormally on 6% SDS-gel electrophoresis in the BB{sub d} rat. S was larger (+5 kDa), D was either smaller (-5 kDa) or unaltered, and AOP was smaller (-5 kDa) in the BB{sub d} than in the normal Wistar. The structural abnormalities were independent of hyperglycemia or ketoacidosis and restored to normal by daily insulin treatment (NPH, 3-4 units/rat) for two to three weeks. Newly-synthesized brush border hydrolases were examined after 6 hours of intraperitoneal injection of ({sup 35}S) methionine (2 mCi) and found to be altered, suggesting that structural abnormality appeared acutely during intracellular synthesis rather than being due to slow extracellular modifications such as non-enzymatic glycosylation. Deglycosylation of brush border proteins by trifluoromethanesulfonic acid resulted in an apoprotein with normal electrophoretic migration in BB{sub d}, indicating that the alteration was due to the carbohydrates component of the glycoprotein. Pulse-chase studies with ({sup 35}S) methionine were consistent with normal protein an co-translational and initial N-linked carbohydrate assembly in association with the endoplasmic reticulum in BB{sub d}. However, the post-translational maturation of N-linked and addition of 0-linked carbohydrate chains in Golgi were prolonged, and produced a larger single-chain precursor of S-D in BB{sub d} than normal.

  8. Alterations of intestinal glycoprotein hydrolases in congenital diabetes

    International Nuclear Information System (INIS)

    Najjar, S.M.

    1989-01-01

    The diabetic BioBreed (BB d ) rat was used for the study of the molecular structure of intestinal brush border sucrase-α-dextrinase (SD) and aminooligopeptidase (AOP) in diabetes mellitus. The specific catalytic activity of S-D and AOP in the BB d rat is normal. However, solid-phase radioimmunoassay revealed loss of some antigenic determinants in the BB d rat. S-D and AOP migrated abnormally on 6% SDS-gel electrophoresis in the BB d rat. S was larger (+5 kDa), D was either smaller (-5 kDa) or unaltered, and AOP was smaller (-5 kDa) in the BB d than in the normal Wistar. The structural abnormalities were independent of hyperglycemia or ketoacidosis and restored to normal by daily insulin treatment (NPH, 3-4 units/rat) for two to three weeks. Newly-synthesized brush border hydrolases were examined after 6 hours of intraperitoneal injection of [ 35 S] methionine (2 mCi) and found to be altered, suggesting that structural abnormality appeared acutely during intracellular synthesis rather than being due to slow extracellular modifications such as non-enzymatic glycosylation. Deglycosylation of brush border proteins by trifluoromethanesulfonic acid resulted in an apoprotein with normal electrophoretic migration in BB d , indicating that the alteration was due to the carbohydrates component of the glycoprotein. Pulse-chase studies with [ 35 S] methionine were consistent with normal protein an co-translational and initial N-linked carbohydrate assembly in association with the endoplasmic reticulum in BB d . However, the post-translational maturation of N-linked and addition of 0-linked carbohydrate chains in Golgi were prolonged, and produced a larger single-chain precursor of S-D in BB d than normal

  9. Increasing nerve agent treatment efficacy by P-glycoprotein inhibition.

    Science.gov (United States)

    Joosen, Marloes J A; Vester, Stefanie M; Hamelink, Jouk; Klaassen, Steven D; van den Berg, Roland M

    2016-11-25

    One of the shortcomings of current treatment of nerve agent poisoning is that not all drugs effectively penetrate the blood-brain barrier (BBB), whereas most nerve agents easily do. P-glycoprotein (Pgp) efflux transporters at the BBB may contribute to this aspect. It was previously shown that Pgp inhibition by tariquidar enhanced the efficacy of nerve agent treatment when administered as a pretreatment. In the present study soman-induced seizures were also substantially prevented when the animals were intravenously treated with tariquidar post-poisoning, in addition to HI-6 and atropine. In these animals, approximately twice as much AChE activity was present in their brain as compared to control rats. The finding that tariquidar did not affect distribution of soman to the brain indicates that the potentiating effects were a result of interactions of Pgp inhibition with drug distribution. In line with this, atropine appeared to be a substrate for Pgp in in vitro studies in a MDR1/MDCK cell model. This indicates that tariquidar might induce brain region specific effects on atropine distribution, which could contribute to the therapeutic efficacy increase found. Furthermore, the therapeutic enhancement by tariquidar was compared to that of the less specific and less potent Pgp inhibitor cyclosporine A. This compound appeared to induce a protective effect similar to tariquidar. In conclusion, treatment with a Pgp inhibitor resulted in enhanced therapeutic efficacy of HI-6 and atropine in a soman-induced seizure model in the rat. The mechanism underlying these effects should be further investigated. To that end, the potentiating effect of nerve agent treatment should be addressed against a broader range of nerve agents, for oximes and atropine separately, and for those at lower doses. In particular when efficacy against more nerve agents is shown, a Pgp inhibitor such as tariquidar might be a valid addition to nerve agent antidotes. Copyright © 2016 Elsevier Ireland

  10. Membrane topology analysis of HIV-1 envelope glycoprotein gp41

    Directory of Open Access Journals (Sweden)

    Xiao Dan

    2010-11-01

    Full Text Available Abstract Background The gp41 subunit of the HIV-1 envelope glycoprotein (Env has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD. An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP that is only active in the cytoplasm. The tag protein (HaloTag and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system. Results In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells, the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability. Conclusions It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.

  11. Effect of expression of P-glycoprotein on technetium-99m methoxyisobutylisonitrile single photon emission computed tomography of brain tumors

    Energy Technology Data Exchange (ETDEWEB)

    Shibata, Yasushi; Matsumura, Akira; Nose, Tadao [Tsukuba Univ., Ibaraki (Japan). Inst. of Clinical Medicine

    2002-08-01

    The expression of P-glycoprotein was investigated imunohistochemically in 26 brain tumor tissues and compared with the findings of technetium-99m methoxyisobutylisonitrile single photon emission computed tomography ({sup 99m}Tc-MIBI SPECT) to clarify the effect of P-glycoprotein on the diagnostic accuracy. P-glycoprotein labeling index of both tumor cells and vascular endothelial cells showed no clear relationship with the findings of {sup 99m}Tc-MIBI SPECT imaging. Expression of P-glycoprotein has no effect on the diagnostic accuracy of {sup 99m}Tc-MIBI SPECT. (author)

  12. Spatial localization of the Ebola virus glycoprotein mucin-like domain determined by cryo-electron tomography.

    Science.gov (United States)

    Tran, Erin E H; Simmons, James A; Bartesaghi, Alberto; Shoemaker, Charles J; Nelson, Elizabeth; White, Judith M; Subramaniam, Sriram

    2014-09-01

    The Ebola virus glycoprotein mucin-like domain (MLD) is implicated in Ebola virus cell entry and immune evasion. Using cryo-electron tomography of Ebola virus-like particles, we determined a three-dimensional structure for the full-length glycoprotein in a near-native state and compared it to that of a glycoprotein lacking the MLD. Our results, which show that the MLD is located at the apex and the sides of each glycoprotein monomer, provide a structural template for analysis of MLD function. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Bioactivity of proteins isolated from Lactobacillus plantarum L67 treated with Zanthoxylum piperitum DC glycoprotein.

    Science.gov (United States)

    Song, S; Oh, S; Lim, K-T

    2015-06-01

    Lactobacilli in the human gastrointestinal tract have beneficial effects on the health of their host. To enhance these effects, the bioactivity of lactobacilli can be fortified through exogenous dietary or pharmacological agents, such as glycoproteins. To elucidate the inductive effect of Zanthoxylum piperitum DC (ZPDC) glycoprotein on Lactobacillus plantarum L67, we evaluated the radical-scavenging activity, anti-oxidative enzymes (SOD, GPx and CAT), growth rate, ATPase activity and β-galactosidase activity of this strain. When Lact. plantarum L67 was treated with ZPDC glycoprotein at different concentrations, the intensities of a few SDS-PAGE bands were slightly changed. The amount of a 23 kDa protein was increased upon treatment with increasing concentrations of ZPDC glycoprotein. The results of this study indicate that the radical-scavenging activity for O2(-) and OH¯, but not for the DPPH radical, increased in a concentration-dependent manner after treatment with ZPDC glycoprotein. The activation of anti-oxidative enzymes (SOD, GPx and CAT), growth rate and β-galactosidase activity also increased in a concentration-dependent manner in response to ZPDC glycoprotein treatment, whereas ATPase activity was decreased. In summary, ZPDC glycoprotein stimulated an increase in the bioactivity of Lact. plantarum L67. Significance and impact of the study: This study demonstrated that Lactobacillus plantarum L67 possesses anti-oxidative activity. This strain of lactic bacteria has been known to have various probiotic uses, such as yogurt starters and dietary additional supplements. We found, through this experiment, that the protein has a strong anti-oxidative character, and the activity can be enhanced by treatment with Zanthoxylum piperitum DC (ZPDC) glycoprotein. This study may be application of Lact. plantarum L67 treated by ZPDC glycoprotein in yogurt fermentation. It could be one of the avenues of minimizing yogurt postacidification during storage. In addition

  14. Characterization of multidrug resistance P-glycoprotein transport function with an organotechnetium cation

    Energy Technology Data Exchange (ETDEWEB)

    Piwnica-Worms, D.; Vallabhaneni, V.R. [Washington Univ. Medical School, St. Louis, MO (United States); Kronauge, J.F. [Harvard Medical School, Boston, MA (United States)] [and others

    1995-09-26

    Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an {approximately}170 integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis(2-methoxyisobutyl isonitrile) technetium(I) (Tc-SESTAMIBI), a {gamma}-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate. Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulation (T{sub 1/2} {approx} 6 min) of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation. 70 refs., 7 figs., 2 tabs.

  15. The expression and serological reactivity of recombinant canine herpesvirus 1 glycoprotein D

    Directory of Open Access Journals (Sweden)

    MarkéŽta Vaňkov‡á

    2016-01-01

    Full Text Available The aim of this work was to express recombinant glycoprotein D of canine herpesvirus 1 in bacterial cells and to evaluate its diagnostic sensitivity and specificity when compared to traditional serological methods. The gene fragment coding glycoprotein D of canine herpesvirus 1 was amplified by polymerase chain reaction, cloned into plasmid vector and expressed in Escherichia coli cells. Recombinant protein was then purified and used as an antigen in immunoblot for a detection of canine herpesvirus 1 specific antibodies. Antibody testing was performed on the panel of 100 canine sera by immunoblot with recombinant glycoprotein D as antigen and compared with indirect immunofluorescence assay. Serum samples were collected from 83 dogs with no history of canine herpesvirus 1 or reproductive disorders, and from 17 dogs from breeding kennels with a history of canine herpesvirus 1 related reproductive disorders. Sensitivity of glycoprotein D based immunoblot was 89.2% and specificity was 93%. Kappa value was calculated to be 0.8 between immunoblot and indirect immunofluorescence assay. Antibodies against canine herpesvirus 1 infection were detected in 33% of samples by immunoblot assay. Our study confirms that recombinant glycoprotein D expressed in bacterial cells could be used as a suitable and sensitive antigen for immunological tests and that herpesvirus infection seems to be common among the canine population in the Czech Republic.

  16. Guidelines for cloning, expression, purification and functional characterization of primary HIV-1 envelope glycoproteins.

    Science.gov (United States)

    Benureau, Yann; Colin, Philippe; Staropoli, Isabelle; Gonzalez, Nuria; Garcia-Perez, Javier; Alcami, Jose; Arenzana-Seisdedos, Fernando; Lagane, Bernard

    2016-10-01

    The trimeric HIV-1 envelope (Env) glycoproteins gp120 and gp41 mediate virus entry into target cells by engaging CD4 and the coreceptors CCR5 or CXCR4 at the cell surface and driving membrane fusion. Receptor/gp120 interactions regulate the virus life cycle, HIV infection transmission and pathogenesis. Env is also the target of neutralizing antibodies. Efforts have thus been made to produce soluble HIV-1 glycoproteins to develop vaccines and study the role and mechanisms of HIV/receptor interactions. However, production and purification of Env glycoproteins and their functional assessment has to cope with multiple obstacles. These include difficulties in amplifying and cloning env sequences and setting up receptor binding assays that are suitable for studies on large collections of glycoproteins, flexible enough to adapt to Env and receptor structural heterogeneities, and allow recapitulating the receptor binding properties of virion-associated Env trimers. Here we identify these difficulties and present protocols to produce primary gp120 and determination of their binding properties to receptors. The receptor binding assays confirmed that the produced glycoproteins are competent for binding CD4 and undergo proper CD4-induced conformational changes required for interaction with CCR5. These assays may help elucidate the role of gp120/receptor interactions in the pathophysiology of HIV infection and develop HIV-1 entry inhibitors. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Co-treatment by docetaxel and vinblastine breaks down P-glycoprotein mediated chemo-resistance

    Directory of Open Access Journals (Sweden)

    Mahsa Mohseni

    2016-03-01

    Results: Combination treatment of the cells with docetaxel and vinblastine decreased the IC50 values for docetaxel from (30±3.1 to (15±2.6 nM and for vinblastine from (30±5.9 to (5±5.6 nM (P≤0.05.               P-glycoprotein mRNA expression level showed a significant up-regulation in the cells incubated with each drug alone (P≤0.001. Incubation of the cells with combined concentrations of both agents neutralized P-glycoprotein overexpression (P≤0.05. Adding verapamil, a P-glycoprotein inhibitor caused a further increase in the percentage of apoptotic cells when the cells were treated with both agents.  Conclusion:Our results suggest that combination therapy along with P-glycoprotein inhibition can be considered as a novel approach to improve the efficacy of chemotherapeutics in cancer patients with high P-glycoprotein expression.

  18. Distinct requirements for signal peptidase processing and function in the stable signal peptide subunit of the Junin virus envelope glycoprotein

    International Nuclear Information System (INIS)

    York, Joanne; Nunberg, Jack H.

    2007-01-01

    The arenavirus envelope glycoprotein (GP-C) retains a cleaved and stable signal peptide (SSP) as an essential subunit of the mature complex. This 58-amino-acid residue peptide serves as a signal sequence and is additionally required to enable transit of the assembled GP-C complex to the Golgi, and for pH-dependent membrane fusion activity. We have investigated the C-terminal region of the Junin virus SSP to study the role of the cellular signal peptidase (SPase) in generating SSP. Site-directed mutagenesis at the cleavage site (positions - 1 and - 3) reveals a pattern of side-chain preferences consistent with those of SPase. Although position - 2 is degenerate for SPase cleavage, this residue in the arenavirus SSP is invariably a cysteine. In the Junin virus, this cysteine is not involved in disulfide bonding. We show that replacement with alanine or serine is tolerated for SPase cleavage but prevents the mutant SSP from associating with GP-C and enabling transport to the cell surface. Conversely, an arginine mutation at position - 1 that prevents SPase cleavage is fully compatible with GP-C-mediated membrane fusion activity when the mutant SSP is provided in trans. These results point to distinct roles of SSP sequences in SPase cleavage and GP-C biogenesis. Further studies of the unique structural organization of the GP-C complex will be important in identifying novel opportunities for antiviral intervention against arenaviral hemorrhagic disease

  19. Blocking Antibody Access to Neutralizing Domains on Glycoproteins Involved in Entry as a Novel Mechanism of Immune Evasion by Herpes Simplex Virus Type 1 Glycoproteins C and E▿

    Science.gov (United States)

    Hook, Lauren M.; Huang, Jialing; Jiang, Ming; Hodinka, Richard; Friedman, Harvey M.

    2008-01-01

    Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) blocks complement activation, and glycoprotein E (gE) interferes with IgG Fc-mediated activities. While evaluating gC- and gE-mediated immune evasion in human immunodeficiency virus (HIV)-HSV-1-coinfected subjects, we noted that antibody alone was more effective at neutralizing a strain with mutations in gC and gE (gC/gE) than a wild-type (WT) virus. This result was unexpected since gC and gE are postulated to interfere with complement-mediated neutralization. We used pooled human immunoglobulin G (IgG) from HIV-negative donors to confirm the results and evaluated mechanisms of the enhanced antibody neutralization. We demonstrated that differences in antibody neutralization cannot be attributed to the concentrations of HSV-1 glycoproteins on the two viruses or to the absence of an IgG Fc receptor on the gC/gE mutant virus or to enhanced neutralization of the mutant virus by antibodies that target only gB, gD, or gH/gL, which are the glycoproteins involved in virus entry. Since sera from HIV-infected subjects and pooled human IgG contain antibodies against multiple glycoproteins, we determined whether differences in neutralization become apparent when antibodies to gB, gD, or gH/gL are used in combination. Neutralization of the gC/gE mutant was greatly increased compared that of WT virus when any two of the antibodies against gB, gD, or gH/gL were used in combination. These results suggest that gC and gE on WT virus provide a shield against neutralizing antibodies that interfere with gB-gD, gB-gH/gL, or gD-gH/gL interactions and that one function of virus neutralization is to prevent interactions between these glycoproteins. PMID:18480440

  20. Therapeutic vaccine using a monoclonal antibody against a 70-kDa glycoprotein in mice infected with highly virulent Sporothrix schenckii and Sporothrix brasiliensis.

    Science.gov (United States)

    de Almeida, José Roberto Fogaça; Kaihami, Gilberto Hideo; Jannuzzi, Grasielle Pereira; de Almeida, Sandro Rogerio

    2015-01-01

    Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungi that comprise the Sporothrix complex. The latter are widely distributed in nature, developing a saprophytic mycelial form on plant debris and soil. Formerly, the S. schenckii species was thought to be the only species capable of causing sporotrichosis. However, in recent years, the existence of a group of highly genotypically and phenotypically variable species has been reported as etiologic agents of this mycosis. Recently, it has become important to study aspects such as virulence and the immune response against key members of the Sporothrix complex and to observe the presence of glycoprotein (gp) 70 and efficacy of the P6E7 monoclonal antibody against more virulent strains. The data presented here demonstrate that the strain isolated from a case of feline sporotrichosis, that is, strain 5110 (American Type Culture Collection MYA-4823) is the most virulent and the only one able to secrete gp70. This glycoprotein is apparently an important factor in the virulence of Sporothrix spp. because treatment with MAb P6E7 resulted in the reduction of fungal burden in the analyzed organs. Additional studies of the role of gp70 in modulating the immune response of the host are needed to understand the pathology of sporotrichosis. © The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Comparative Analysis of Whey N-Glycoproteins in Human Colostrum and Mature Milk Using Quantitative Glycoproteomics.

    Science.gov (United States)

    Cao, Xueyan; Song, Dahe; Yang, Mei; Yang, Ning; Ye, Qing; Tao, Dongbing; Liu, Biao; Wu, Rina; Yue, Xiqing

    2017-11-29

    Glycosylation is a ubiquitous post-translational protein modification that plays a substantial role in various processes. However, whey glycoproteins in human milk have not been completely profiled. Herein, we used quantitative glycoproteomics to quantify whey N-glycosylation sites and their alteration in human milk during lactation; 110 N-glycosylation sites on 63 proteins and 91 N-glycosylation sites on 53 proteins were quantified in colostrum and mature milk whey, respectively. Among these, 68 glycosylation sites on 38 proteins were differentially expressed in human colostrum and mature milk whey. These differentially expressed N-glycoproteins were highly enriched in "localization", "extracellular region part", and "modified amino acid binding" according to gene ontology annotation and mainly involved in complement and coagulation cascades pathway. These results shed light on the glycosylation sites, composition and biological functions of whey N-glycoproteins in human colostrum and mature milk, and provide substantial insight into the role of protein glycosylation during infant development.

  2. Characterization of Intact Neo-Glycoproteins by Hydrophilic Interaction Liquid Chromatography

    Directory of Open Access Journals (Sweden)

    Alice Pedrali

    2014-06-01

    Full Text Available In this study, an HPLC HILIC-UV method was developed for the analysis of intact neo-glycoproteins. During method development the experimental conditions evaluated involved different HILIC columns (TSKgel Amide-80 and ZIC-pHILIC, and water-acetonitrile mixtures containing various types of acids and salts. The final selected method was based on a TSKgel Amide-80 column and a mobile phase composed of acetonitrile and water both containing 10 mM HClO4. The influence of temperature and sample preparation on the chromatographic performances of the HILIC method was also investigated. The method was applied to the separation of neo-glycoproteins prepared starting from the model protein RNase A by chemical conjugation of different glycans. Using the method here reported it was possible to monitor by UV detection the glycosylation reaction and assess the distribution of neo-glycoprotein isoforms without laborious sample workup prior to analysis.

  3. Contribution of the attachment G glycoprotein to pathogenicity and immunogenicity of avian metapneumovirus subgroup C.

    Science.gov (United States)

    Govindarajan, Dhanasekaran; Kim, Shin-Hee; Samal, Siba K

    2010-03-01

    Avian metapneumovirus (AMPV) causes an upper respiratory tract infection in turkeys leading to serious economic losses to the turkey industry. The G glycoprotein of AMPV is known to be associated with viral attachment and pathogenesis. In this study, we determined the role of the G glycoprotein in the pathogenicity and immunogenicity of AMPV strain Colorado (AMPV/CO). Recombinant AMPV/CO lacking the G protein (rAMPV/CO-deltaG) was generated using a reverse-genetics system. The recovered rAMPV/CO-deltaG replicated slightly better than did wild-type AMPV in Vero cells. However, deletion of the G gene in AMPV resulted in attenuation of the virus in turkeys. The mutant virus induced less-severe clinical signs and a weaker immune response in turkeys than did the wild-type AMPV. Our results suggest that the G glycoprotein is an important determinant for the pathogenicity and immunogenicity of AMPV.

  4. Complement inhibition enables tumor delivery of LCMV glycoprotein pseudotyped viruses in the presence of antiviral antibodies

    Directory of Open Access Journals (Sweden)

    Laura Evgin

    2016-01-01

    Full Text Available The systemic delivery of therapeutic viruses, such as oncolytic viruses or vaccines, is limited by the generation of neutralizing antibodies. While pseudotyping of rhabdoviruses with the lymphocytic choriomeningitis virus glycoprotein has previously allowed for multiple rounds of delivery in mice, this strategy has not translated to other animal models. For the first time, we provide experimental evidence that antibodies generated against the lymphocytic choriomeningitis virus glycoprotein mediate robust complement-dependent viral neutralization via activation of the classical pathway. We show that this phenotype can be capitalized upon to deliver maraba virus pseudotyped with the lymphocytic choriomeningitis virus glycoprotein in a Fischer rat model in the face of neutralizing antibody through the use of complement modulators. This finding changes the understanding of the humoral immune response to arenaviruses, and also describes methodology to deliver viral vectors to their therapeutic sites of action without the interference of neutralizing antibody.

  5. Efficient transduction of neurons using Ross River glycoprotein-pseudotyped lentiviral vectors

    DEFF Research Database (Denmark)

    Jakobsson, J; Nielsen, T Tolstrup; Staflin, K

    2006-01-01

    Lentiviral vectors are promising tools for CNS gene transfer since they efficiently transduce the cells of the nervous system in vivo. In this study, we have investigated the transduction efficiency of lentiviral vectors pseudotyped with Ross River virus glycoprotein (RRV-G) (RRV-G-pseudotyped le......Lentiviral vectors are promising tools for CNS gene transfer since they efficiently transduce the cells of the nervous system in vivo. In this study, we have investigated the transduction efficiency of lentiviral vectors pseudotyped with Ross River virus glycoprotein (RRV-G) (RRV...... and human glial fibrillary acidic protein, we demonstrated cell-specific transgene expression in the desired cell type. Ross River virus glycoprotein-pseudotyped lentiviral vectors also transduced human neural progenitor cells in vitro, showing that receptors for the RRV-G are present on human neural cells....

  6. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    Science.gov (United States)

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism.

  7. Sphingolipid signaling reduces basal P-glycoprotein activity in renal proximal tubule.

    Science.gov (United States)

    Miller, David S

    2014-03-01

    P-glycoprotein is an ATP-driven xenobiotic export pump that is highly expressed in barrier and excretory tissues, where it greatly influences drug pharmacokinetics. Recent studies in the blood-brain and spinal cord barriers identified a sphingolipid-based signaling pathway that regulates basal activity of P-glycoprotein. Here we use an established comparative renal model that permits direct measurement of P-glycoprotein activity to determine whether such signaling occurs in another tissue, killifish renal proximal tubule. Isolated killifish tubules exposed to 0.01-1.0 μM sphingosine-1-phosphate (S1P) exhibited a profound decrease in P-glycoprotein transport activity, measured as specific accumulation of a fluorescent cyclosporine A derivative in the tubule lumen. Loss of activity had a rapid onset and was fully reversible when the S1P was removed. Transport mediated by multidrug resistance-associated protein 2 (Mrp2) or a teleost fish organic anion transporter (Oat) was not affected. S1P effects were blocked by a specific S1P receptor 1 (S1PR1) antagonist and mimicked by a S1PR agonist. Sphingosine also reduced P-glycoprotein transport activity and those effects were blocked by an inhibitor of sphingosine kinase and by the S1PR1 antagonist. These results for a comparative renal model suggest that sphingolipid signaling to P-glycoprotein is not just restricted to the blood-brain and blood-spinal cord barriers, but occurs in other excretory and barrier tissues.

  8. Dopamine stimulates snail albumen gland glycoprotein secretion through the activation of a D1-like receptor.

    Science.gov (United States)

    Mukai, S T; Kiehn, L; Saleuddin, A S M

    2004-06-01

    The catecholamine dopamine is present in both the central nervous system and in the peripheral tissues of molluscs, where it is involved in regulating reproduction. Application of exogenous dopamine to the isolated albumen gland of the freshwater pulmonate snail Helisoma duryi (Wetherby) induces the secretion (release) of perivitelline fluid. The major protein component of the perivitelline fluid of Helisoma duryi is a native 288 kDa glycoprotein that is secreted around individual eggs and serves as an important source of nutrients for the developing embryos. The secretion of glycoprotein by the albumen gland is a highly regulated event that must be coordinated with the arrival of the fertilized ovum at the carrefour (the region where the eggs receive albumen gland secretory products). In order to elucidate the intracellular signalling pathway(s) mediating dopamine-induced glycoprotein secretion, albumen gland cAMP production and glycoprotein secretion were measured in the presence/absence of selected dopamine receptor agonists and antagonists. Dopamine D1-selective agonists dihydrexidine, 6,7-ADTN and SKF81297 stimulated cAMP production and glycoprotein secretion from isolated albumen glands whereas D1-selective antagonists SCH23390 and SKF83566 suppressed dopamine-stimulated cAMP production. Dopamine D2-selective agonists and antagonists generally had no effect on cAMP production or protein secretion. Based on the effects of these compounds, a pharmacological profile was obtained that strongly suggests the presence of a dopamine D1-like receptor in the albumen gland of Helisoma duryi. In addition, secretion of albumen gland glycoprotein was not inhibited by protein kinase A inhibitors, suggesting that dopamine-stimulated protein secretion might occur through a protein kinase A-independent pathway.

  9. Glycoproteins E and I facilitate neuron-to-neuron spread of herpes simplex virus.

    OpenAIRE

    Dingwell, K S; Doering, L C; Johnson, D C

    1995-01-01

    Two herpes simplex virus (HSV) glycoproteins E and I (gE and gI) form a heterooligomer which acts as an Fc receptor and also facilitates cell-to-cell spread of virus in epithelial tissues and between certain cultured cells. By contrast, gE-gI is not required for infection of cells by extracellular virus. HSV glycoproteins gD and gJ are encoded by neighboring genes, and gD is required for both virus entry into cells and cell-to-cell spread, whereas gJ has not been shown to influence these proc...

  10. Assessment of lectin and HILIC based enrichment protocols for characterization of serum glycoproteins by mass spectrometry

    DEFF Research Database (Denmark)

    Calvano, Cosima D; Zambonin, Carlo G; Jensen, Ole Nørregaard

    2008-01-01

    identified using a mixture of three immobilized lectins for consecutive glycoprotein enrichment and glycopeptide enrichment. The combination of lectin affinity enrichment of glycoproteins and subsequent HILIC enrichment of tryptic glycopeptides identified 81 N-glycosylation sites in 44 proteins. A total...... of 63 glycosylation sites in 38 proteins were identified by both methods, demonstrating distinct differences and complementarity. Serial application of custom-made microcolumns of mixed, immobilized lectins proved efficient for recovery and analysis of glycopeptides from serum samples of breast cancer...... patients and healthy individuals to assess glycosylation site frequencies....

  11. High P-glycoprotein-mediated export observed in patients with a history of idiopathic thrombocytopenic purpura.

    Science.gov (United States)

    Levy, Adam S; Cunningham-Rundles, Susanna; Mazza, BethAnne; Simm, Maciej; Gorlick, Richard; Bussel, James

    2002-09-01

    Studies have suggested that high P-glycoprotein expression in lymphocytes from patients with autoimmune disorders may affect disease outcome. Idiopathic thrombocytopenic purpura (ITP) and Evans' syndrome are widely thought to be autoimmune processes, however, the precise mechanisms remain unknown. Peripheral blood mononuclear cells from patients with refractory or recurrent ITP or Evans' syndrome were studied using the rhodamine 123 flow cytometric assay to investigate functional export levels. Lymphocytes from ITP and Evans' syndrome patients showed a significantly decreased ability to retain rhodamine, suggesting increased export protein function. Reverse transcription polymerase chain reaction distinguished P-glycoprotein as the likely export protein.

  12. Hypolipidemic effect and antioxidant activity of glycoprotein isolated from Ulmus davidiana Nakai in Triton WR-1339-treated mouse.

    Science.gov (United States)

    Ko, Jeong-Hyeon; Lee, Sei-Jung; Lim, Kye-Taek

    2007-01-01

    The glycoprotein isolated from Ulmus davidiana Nakai (UDN) (UDN glycoprotein) has a molecular weight of 116 kDa and consists of 78.65% carbohydrate content and 21.35% protein content. In the present study, we investigated the hypolipidemic effect of UDN glycoprotein on Triton WR-1339-induced mice. With pretreatment with UDN glycoprotein, the triacylglycerol (TAG), total cholesterol and low density lipoprotein-cholesterol (LDL-C) concentrations were significantly reduced, whereas high density lipoprotein-cholesterol (HDL-C) concentration was increased in the plasma of Triton WR-1339-induced mice. With respect to antioxidative activity, UDN glycoprotein significantly decreased the level of thiobarbituric acid reactive substances (TBARS) and improved activities of catalase and glutathione peroxidase (GPx), without an apparent change of superoxide dismutase (SOD) activity. Also UDN glycoprotein significantly increased nitric oxide (NO) production in Triton WR-1339-induced mice. These results indicate that UDN glycoprotein has a hypolipidemic effect, possesses antioxidant activity and has an ability to stimulate NO production. Thus, we speculate that UDN glycoprotein is an example of natural compound that lowers plasma lipid level together with having an antioxidant function in Triton WR-1339-induced mice.

  13. Conglutinin binds the HIV-1 envelope glycoprotein gp 160 and inhibits its interaction with cell membrane CD4

    DEFF Research Database (Denmark)

    Andersen, Ove; Sørensen, A M; Svehag, S E

    1991-01-01

    The highly glycosylated envelope glycoprotein (gp 160) of human immunodeficiency virus (HIV) interacts with the CD4 molecule present on the membrane of CD4+ cells and is involved in the pathobiology of HIV infection. Lectins bind glycoproteins through non-covalent interactions with specific hexose...

  14. Synthesis of peptide-immunogens corresponding to amino acid sequences from human histocompatibility class II membrane glycoproteins.

    Science.gov (United States)

    Chillemi, F; Cappelletti, S; Francescato, P; Chersi, A

    1990-03-01

    Six peptides with amino acid sequences of human histocompatibility Class II membrane glycoproteins were synthesized by conventional solution methods. Five peptides were prepared by stepwise procedures from the carboxyterminus. The sixth was synthesized by fragment condensation (5 + 10 coupling). Antibodies to synthetic peptides were then used to locate exposed and buried regions in the membrane glycoproteins.

  15. Appearance and cellular distribution of lectin-like receptors for alpha 1-acid glycoprotein in the developing rat testis

    DEFF Research Database (Denmark)

    Andersen, U O; Bøg-Hansen, T C; Kirkeby, S

    1996-01-01

    A histochemical avidin-biotin technique with three different alpha 1-acid glycoprotein glycoforms showed pronounced alterations in the cellular localization of two alpha 1-acid glycoprotein lectin-like receptors during cell differentiation in the developing rat testis. The binding of alpha 1-acid...

  16. P-glycoprotein interaction with risperidone and 9-OH-risperidone studied in vitro, in knock-out mice and in drug-drug interaction experiments

    DEFF Research Database (Denmark)

    Ejsing, Thomas B.; Pedersen, Anne D.; Linnet, Kristian

    2005-01-01

    P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice......P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice...

  17. EMA: a developmentally regulated cell-surface glycoprotein of CNS neurons that is concentrated at the leading edge of growth cones.

    Science.gov (United States)

    Baumrind, N L; Parkinson, D; Wayne, D B; Heuser, J E; Pearlman, A L

    1992-08-01

    To identify cell-surface molecules that mediate interactions between neurons and their environment during neural development, we used monoclonal antibody techniques to define a developmentally regulated antigen in the central nervous system of the mouse. The antibody we produced (2A1) immunolabels cells throughout the central nervous system; we analyzed its distribution in the developing cerebral cortex, where it is expressed on cells very soon after they complete mitosis and leave the periventricular proliferative zone. Expression continues into adult life. The antibody also labels the epithelium of the choroid plexus and the renal proximal tubules, but does not label neurons of the peripheral nervous system in the dorsal root ganglia. In dissociated cell culture of embryonic cerebral cortex, 2A1 labels the surface of neurons but not glia. Immunolabeling of neurons in tissue culture is particularly prominent on the edge of growth cones, including filopodia and the leading edge of lamellipodia, when observed with either immunofluorescence or freeze-etch immunoelectron microscopy. Immunopurification with 2A1 of a CHAPS-extracted membrane preparation from brains of neonatal mice produces a broad (32-36 kD) electrophoretic band and a less prominent 70 kD band that are sensitive to N-glycosidase but not endoglycosidase H. Thus the 2A1 antibody recognizes a developmentally regulated, neuronal cell surface glycoprotein (or glycoproteins) with complex N-linked oligosaccharide side chains. We have termed the glycoprotein antigen EMA because of its prominence on the edge membrane of growth cones. EMA is similar to the M6 antigen (Lagenaur et al: J. Neurobiol. 23:71-88, 1992) in apparent molecular weight, distribution in tissue sections, and immunoreactivity on Western blots, suggesting that the two antigens are similar or identical. Expression of EMA is a very early manifestation of neuronal differentiation; its distribution on growth cones suggests a role in mediating the

  18. A Novel Method for Detection of Glycoproteins on Sodium Dodecyl Sulphate Polyacrylamide Gel Using Radio-Iodinated Tyrosine

    DEFF Research Database (Denmark)

    Nalla, Amarnadh; Draz, Hossam M.; Dole, Anita

    2009-01-01

    A (Con A) were used as a glycosylated and a non-glycosylated model proteins, respectively. The proteins were separated in SDS- PAGE and oligosaccharides on the glycoprotein were oxidised using periodic acid to produce aldehydes than 125I-tyroine was conjugated to aldehyde groups without using reducing...... agent like Sodium Metabisulfite. The radio-iodinated glycoprotein on gel was scanned using a Multi-Photon Detection (MPD) scanner. The elechtrophoretic analysis of ovalbumin and Con A were performed and stained with Coomassie brilliant blue to identify total proteins, while MPD detection...... of glycoproteins using 125I-tyrosine selectively detected ovalbumin. Present results showed that MPD enhanced glycoprotein detection method can be used as a sensitive tool for the detection of glycoproteins on polyacrylamide gel...

  19. Chemoenzymatic site-specific labeling of influenza glycoproteins as a tool to observe virus budding in real time.

    Directory of Open Access Journals (Sweden)

    Maximilian Wei-Lin Popp

    Full Text Available The influenza virus uses the hemagglutinin (HA and neuraminidase (NA glycoproteins to interact with and infect host cells. While biochemical and microscopic methods allow examination of the early steps in flu infection, the genesis of progeny virions has been more difficult to follow, mainly because of difficulties inherent in fluorescent labeling of flu proteins in a manner compatible with live cell imaging. We here apply sortagging as a chemoenzymatic approach to label genetically modified but infectious flu and track the flu glycoproteins during the course of infection. This method cleanly distinguishes influenza glycoproteins from host glycoproteins and so can be used to assess the behavior of HA or NA biochemically and to observe the flu glycoproteins directly by live cell imaging.

  20. Expression of variable viruses as herpes simplex glycoprotein D and varicella zoster gE glycoprotein using a novel plasmid based expression system in insect cell

    Directory of Open Access Journals (Sweden)

    A.M. Al-Sulaiman

    2017-11-01

    Full Text Available Several prokaryotic and eukaryotic expression systems have been used for in vitro production of viruses’ proteins. However eukaryotic expression system was always the first choice for production of proteins that undergo post-translational modification such as glycosylation. Recombinant baculoviruses have been widely used as safe vectors to express heterologous genes in the culture of insect cells, but the manipulation involved in creating, titrating, and amplifying viral stocks make it time consuming and laborious. Therefore, to facilitate rapid expression in insect cell, a plasmid based expression system was used to express herpes simplex type 1 glycoprotein D (HSV-1 gD and varicella zoster glycoprotein E (VZV gE. Recombinant plasmids were generated, transfected into insect cells (SF9, and both glycoproteins were expressed 48 h post-infection. A protein with approximately molecular weight of 64-kDa and 98-kDa for HSV-1 gD and VZV gE respectively was expressed and confirmed by SDS. Proteins were detected in insect cells cytoplasm and outer membrane by immunofluorescence. The antigenicity and immunoreactivity of each protein were confirmed by immunoblot and ELISA. Results suggest that this system can be an alternative to the traditional baculovirus expression for small scale expression system in insect cells.

  1. Comparison of in vitro assays in selecting radiotracers for in vivo P-glycoprotein PET imaging

    NARCIS (Netherlands)

    Raaphorst, R.M.; Savolainen, H.; Cantore, M.; Steeg, E. van de; Waarde, A. van; Colabufo, N.A.; Elsinga, P.H.; Lammertsma, A.A.; Windhorst, A.D.; Luurtsema, G.

    2017-01-01

    Positron emission tomography (PET) imaging of P-glycoprotein (P-gp) in the blood-brain barrier can be important in neurological diseases where P-gp is affected, such as Alzheimer’s disease. Radiotracers used in the imaging studies are present at very small, nanomolar, concentration, whereas in vitro

  2. Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein

    NARCIS (Netherlands)

    Konings, WN; Poelarends, GJ

    2002-01-01

    Most ATP-binding cassette (ABC) multidrug transporters known to date are of eukaryotic origin, such as the P-glycoproteins (Pgps) and multidrug resistance-associated proteins (MRPs). Only one well-characterized ABC multidrug transporter, LmrA, is of bacterial origin. On the basis of its structural

  3. What Do Chaotrope-Based Avidity Assays for Antibodies to HIV-1 Envelope Glycoproteins Measure?

    NARCIS (Netherlands)

    Alexander, Marina R.; Ringe, Rajesh; Sanders, Rogier W.; Voss, James E.; Moore, John P.; Klasse, Per Johan

    2015-01-01

    When HIV-1 vaccine candidates that include soluble envelope glycoproteins (Env) are tested in humans and other species, the resulting antibody responses to Env are sifted for correlates of protection or risk. One frequently used assay measures the reduction in antibody binding to Env antigens by an

  4. The combination of simple MALDI matrices for the improvement of intact glycoproteins and glycans analysis

    Czech Academy of Sciences Publication Activity Database

    Laštovičková, Markéta; Chmelík, Josef; Bobálová, Janette

    2009-01-01

    Roč. 281, 1-2 (2009), s. 82-88 ISSN 1387-3806 R&D Projects: GA AV ČR IAA600040701; GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : glycoproteins * binary matrices * MALDI-TOF MS Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.117, year: 2009

  5. Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

    NARCIS (Netherlands)

    Ringe, Rajesh P.; Sanders, Rogier W.; Yasmeen, Anila; Kim, Helen J.; Lee, Jeong Hyun; Cupo, Albert; Korzun, Jacob; Derking, Ronald; van Montfort, Thijs; Julien, Jean-Philippe; Wilson, Ian A.; Klasse, Per Johan; Ward, Andrew B.; Moore, John P.

    2013-01-01

    We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp) 140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120-gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a

  6. Glycoproteins 66 and 69 kDa of pollen tube wall: properties and distribution in angiosperms

    Czech Academy of Sciences Publication Activity Database

    Fidlerová, A.; Smýkal, P.; Tupý, Jaroslav; Čapková, Věra

    2001-01-01

    Roč. 158, - (2001), s. 1367-1374 ISSN 0176-1617 R&D Projects: GA ČR GA304/00/1622 Institutional research plan: CEZ:AV0Z5038910 Keywords : angiosperms * cell wall * glycoproteins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.018, year: 2001

  7. Human CRISP-3 binds serum alpha(1)B-glycoprotein across species

    DEFF Research Database (Denmark)

    Udby, Lene; Johnsen, Anders H; Borregaard, Niels

    2010-01-01

    CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular...

  8. Structural analysis of the carbohydrate chains of glycoproteins by 500-MHz 1H-NMR spectroscopy

    International Nuclear Information System (INIS)

    Mutsaers, J.H.G.M.

    1986-01-01

    This thesis deals with the structural analysis by 500-MHz 1 H-NMR spectroscopy of carbohydrate chains obtained from glycoproteins. In the chapters 1 to 6 the structural analysis of N-glycosidically linked carbohydrate chains is described. The chapters 7 to 10 describe the structural analysis of O-glycosidically linked carbohydrate chains. 381 refs.; 44 figs.; 24 tabs.; 7 schemes

  9. A high boronate avidity monolithic capillary for the selective enrichment of trace glycoproteins.

    Science.gov (United States)

    Li, Daojin; Li, Yang; Li, Xinglin; Bie, Zijun; Pan, Xianghua; Zhang, Qian; Liu, Zhen

    2015-03-06

    Boronate affinity materials, as effective sample enrichment sorbents for glycoproteomic analysis, have attracted increasing attention in recent years. However, most of boronate affinity materials suffer from an apparent limitation, limited binding strength. As a result, extraction of glycoproteins of trace concentration is rather difficult or impossible. In this study, we present a high boronate avidity monolithic capillary. Branched polyethyleneimine (PEI) was used as a scaffold to amplify the number of boronic acid moieties. While 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA), which exhibited ultrahigh affinity toward cis-diol-containing compounds, was employed as an affinity ligand. Due to the PEI-assisted synergistic multivalent binding, the monolithic column exhibited high boronate avidity toward glycoproteins, with binding constants of 10(-6)-10(-7)M. Such binding strength was the highest among already reported boronic acid-functionalized materials that can be used for glycoproteomic analysis. Besides, the boronate avidity monolithic column exhibited one additional beneficial feature, lowered binding pH (≥6.5). These features greatly favored the selective enrichment of trace glycoproteins from real samples. The feasibility for practical applications was demonstrated with the selective enrichment of trace glycoproteins in human saliva. As compared with other boronate avidity/affinity materials, the boronate avidity monolithic capillary exhibited the best performance. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. St. John's Wort constituents modulate P-glycoprotein transport activity at the blood-brain barrier.

    NARCIS (Netherlands)

    Ott, M.; Huls, M.; Cornelius, M.G.; Fricker, G.

    2010-01-01

    PURPOSE: The purpose of this study was to investigate the short-term signaling effects of St. John's Wort (SJW) extract and selected SJW constituents on the blood-brain barrier transporter P-glycoprotein and to describe the role of PKC in the signaling. METHODS: Cultured porcine brain capillary

  11. P-glycoprotein-deficient mice have proximal tubule dysfunction but are protected against ischemic renal injury

    NARCIS (Netherlands)

    Huls, M.; Kramers, C.; Levtchenko, E.N.; Wilmer, M.J.G.; Dijkman, H.B.P.M.; Kluijtmans, L.A.J.; Hoorn, J.W.A. van der; Russel, F.G.M.; Masereeuw, R.

    2007-01-01

    The multidrug resistance gene 1 product, P-glycoprotein (P-gp), is expressed in several excretory organs, including the apical membrane of proximal tubules. After inducing acute renal failure, P-gp expression is upregulated and this might be a protective function by pumping out toxicants and harmful

  12. Structure of Acidic pH Dengue Virus Showing the Fusogenic Glycoprotein Trimers

    NARCIS (Netherlands)

    Zhang, Xinzheng; Sheng, Ju; Austin, S. Kyle; Hoornweg, Tabitha E.; Smit, Jolanda M.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G.

    Flaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions, the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate viral fusion and nucleocapsid penetration into the cytoplasm. However,

  13. N-glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

    DEFF Research Database (Denmark)

    Pedersen, Carina T; Loke, Ian; Lorentzen, Andrea

    2017-01-01

    Studies of protein N-glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of on...

  14. Characterization of the Outer Domain of the gp120 Glycoprotein from Human Immunodeficiency Virus Type 1

    Science.gov (United States)

    Yang, Xinzhen; Tomov, Vesko; Kurteva, Svetla; Wang, Liping; Ren, Xinping; Gorny, Miroslaw K.; Zolla-Pazner, Susan; Sodroski, Joseph

    2004-01-01

    The core of the gp120 glycoprotein from human immunodeficiency virus type 1 (HIV-1) is comprised of three major structural domains: the outer domain, the inner domain, and the bridging sheet. The outer domain is exposed on the HIV-1 envelope glycoprotein trimer and contains binding surfaces for neutralizing antibodies such as 2G12, immunoglobulin G1b12, and anti-V3 antibodies. We expressed the outer domain of HIV-1YU2 gp120 as an independent protein, termed OD1. OD1 efficiently bound 2G12 and a large number of anti-V3 antibodies, indicating its structural integrity. Immunochemical studies with OD1 indicated that antibody responses against the outer domain of the HIV-1 gp120 envelope glycoprotein are rare in HIV-1-infected human sera that potently neutralize the virus. Surprisingly, such outer-domain-directed antibody responses are commonly elicited by immunization with recombinant monomeric gp120. Immunization with soluble, stabilized HIV-1 envelope glycoprotein trimers elicited antibody responses that more closely resembled those in the sera of HIV-1-infected individuals. These results underscore the qualitatively different humoral immune responses elicited during natural infection and after gp120 vaccination and help to explain the failure of gp120 as an effective vaccine. PMID:15542649

  15. Histidine-rich glycoprotein promotes macrophage activation and inflammation in chronic liver disease

    NARCIS (Netherlands)

    Bartneck, M.; Fech, V.; Ehling, J.; Govaere, O.; Warzecha, K.T.; Hittatiya, K.; Vucur, M.; Gautheron, J.; Luedde, T.; Trautwein, C.; Lammers, Twan Gerardus Gertudis Maria; Roskams, T.; Jahnen-Dechent, W.; Tacke, F.

    2016-01-01

    Pathogen- and injury-related danger signals as well as cytokines released by immune cells influence the functional differentiation of macrophages in chronic inflammation. Recently, the liver-derived plasma protein, histidine-rich glycoprotein (HRG), was demonstrated, in mouse tumor models, to

  16. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei

    NARCIS (Netherlands)

    Zomerdijk, J. C.; Ouellette, M.; ten Asbroek, A. L.; Kieft, R.; Bommer, A. M.; Clayton, C. E.; Borst, P.

    1990-01-01

    The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site

  17. The bacteria binding glycoprotein salivary agglutinin (SAG/gp340) activates complement via the lectin pathway

    NARCIS (Netherlands)

    Leito, Jelani T. D.; Ligtenberg, Antoon J. M.; van Houdt, Michel; van den Berg, Timo K.; Wouters, Diana

    2011-01-01

    Salivary agglutinin (SAG), also known as gp-340 and Deleted in Malignant Brain Tumours 1, is a glycoprotein that is present in tears, lung fluid and mucosal surfaces along the gastrointestinal tract. It is encoded by the Deleted in Malignant Brain Tumours 1 gene, a member of the Scavenger Receptor

  18. Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections

    NARCIS (Netherlands)

    Xiong, Xiaoli; Tortorici, M Alejandra; Snijder, Joost|info:eu-repo/dai/nl/338018328; Yoshioka, Craig; Walls, Alexandra C; Li, Wentao|info:eu-repo/dai/nl/411296272; McGuire, Andrew T; Rey, Félix A; Bosch, Berend-Jan|info:eu-repo/dai/nl/273306049; Veesler, David

    2017-01-01

    Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle-East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to

  19. Patient-derived monoclonal antibodies directed towards beta2 glycoprotein-1 display lupus anticoagulant activity

    NARCIS (Netherlands)

    Dienava-Verdoold, I.; Boon-Spijker, M. G.; de Groot, P. G.; Brinkman, H. J. M.; Voorberg, J.; Mertens, K.; Derksen, R. H. W. M.; de Laat, B.

    2011-01-01

    Patients with antiphospholipid syndrome (APS) display a heterogeneous population of antibodies with beta(2) glycoprotein-1 (β(2)GP1) as the major antigen. We isolated and characterized human mAbs directed against β(2)GP1 from the immune repertoire of APS patients. Variable heavy chain repertoires

  20. Plasma Krebs von den Lungen glycoprotein, lung injury, and noninvasive ventilation in Duchenne muscular dystrophy.

    Science.gov (United States)

    Hamada, Satoshi; Ishikawa, Yuka; Aoyagi, Tomoyuki; Ishikawa, Yukitoshi; Minami, Ryoji; Bach, John R

    2012-10-01

    There have been few reports of ventilator-induced lung injury associated with noninvasive ventilation (NIV), but many with invasive mechanical ventilation. The purpose of this study was to detect subclinical NIV-associated lung injury by monitoring Krebs von den Lungen glycoprotein plasma levels. Forty-one Duchenne muscular dystrophy patients were divided into three categories: group 1, asymptomatic and not using ventilators; group 2, NIV use less than 24 hrs/day at full ventilatory support settings; and group 3, continuous NIV dependence. Plasma Krebs von den Lungen glycoprotein level was measured by electrochemical luminescent immunoassay using Krebs von den Lungen glycoprotein antibodies. One-way analysis of variance, followed by the Tukey-Kramer test, was used as appropriate to compare intergroup differences. Extent of ventilator dependence correlated with age (P Krebs von den Lungen glycoprotein levels were not significantly different. NIV used at volumes and pressures of full (invasive) ventilatory support may not induce the alveolar septal barrier injury commonly seen with invasive mechanical ventilation.

  1. Eosinophil derived neurotoxin (EDN) levels in commercial human urinary preparations of glycoprotein hormones

    NARCIS (Netherlands)

    Kauffman, HF; Hovenga, H; de Bruijn, HWA; Beintema, JJ

    Eosinophil derived neurotoxin (EDN) is a ubiquitous human ribonuclease, occurring not only in eosinophils, but also in many tissues and body fluids. It may be a contaminant of commercial human urinary preparations of chorionic gonadotropin (hCG) and other glycoprotein hormones. Here we describe the

  2. Human intestinal P-glycoprotein activity estimated by the model substrate digoxin

    DEFF Research Database (Denmark)

    Larsen, U L; Hyldahl Olesen, L; Nyvold, Charlotte Guldborg

    2007-01-01

    P-glycoprotein (Pgp) plays a part in the intestinal uptake of xenobiotics and has been associated with susceptibility to ulcerative colitis. The aim of this study was to examine Pgp activity in relation to age, gender, medical treatment (rifampicin or ketoconazole) and the multidrug resistance (MDR...

  3. Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

    DEFF Research Database (Denmark)

    Nieswandt, B; Brakebusch, C; Bergmeier, W

    2001-01-01

    subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed...

  4. Splice variation in the cytoplasmic domains of myelin oligodendrocyte glycoprotein affects its cellular localisation and transport.

    Science.gov (United States)

    Boyle, Louise H; Traherne, James A; Plotnek, Gemma; Ward, Rosemary; Trowsdale, John

    2007-09-01

    Although myelin oligodendrocyte glycoprotein is a candidate autoantigen in multiple sclerosis, its function remains unknown. In humans, mRNA expressed by the myelin oligodendrocyte glycoprotein gene is alternatively spliced resulting in at least nine unique protein isoforms. In this study, we investigated the sub-cellular localisation and membrane trafficking of six isoforms by cloning them into mammalian expression vectors. Confocal microscopy revealed that these protein products are expressed in different cellular compartments. While two full-length isoforms (25.6 and 25.1) are expressed at the cell surface, three alternatively spliced forms (22.7, 21.0 and 20.5) have a more intracellular distribution, localising to the endoplasmic reticulum and/or endosomes. Isoform 16.3, which lacks a transmembrane domain, is secreted. A switch in the sub-cellular localisation of myelin oligodendrocyte glycoprotein may have profound effects on receptor:ligand interactions and consequently the function of the protein. The structural features of the alternative isoforms and their differential, sub-cellular expression patterns could dictate the exposure of major immunogenic determinants within the central nervous system. Our findings highlight myelin oligodendrocyte glycoprotein splicing as a factor that could be critical to the phenotypic expression of multiple sclerosis.

  5. Expression and structural-functional alterations of α-1-acid glycoprotein at the pathological state

    Directory of Open Access Journals (Sweden)

    Kulinich A. O.

    2010-07-01

    Full Text Available The review analyzes up-to-date knowledge on structure and biological functions of α-acid glycoprotein. The special attention is given to alterations of fucosylation, sialylation and branching of orosomucoid at the acute, chronic inflammation and oncotransformations.

  6. Blood-Brain Barrier P-Glycoprotein Function in Neurodegenerative Disease

    NARCIS (Netherlands)

    Bartels, A. L.

    Protection of the brain is strengthened by active transport and ABC transporters. P-glycoprotein (P-gp) at the blood-brain barrier (BBB) functions as an active efflux pump by extruding a substrate from the brain, which is important for maintaining loco-regional homeostasis in the brain and

  7. Structure of three acidic O-linked carbohydrate chains of porcine zona pellucida glycoproteins

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Hokke, C.H.; Damm, J.B.L.; Kamerling, J.P.

    1993-01-01

    Structural analysis by ID and 2D 1H NMR spectroscopy of three acidic O-linked oligosaccharide alditols, released from porcine zona pellucida glycoproteins by alkaline borohydride treatment, afforded the following structures: Gal beta l-4(6SO4-)GlcNAc beta l-3Gal beta l-4GlcNAc beta 1-3Gal beta

  8. Development of Recombinant Newcastle Disease Viruses Expressing the Glycoprotein (G) of Avian Metapneumovirus as Bivalent Vaccines

    Science.gov (United States)

    Using reverse genetics technology, Newcastle disease virus (NDV) LaSota strain-based recombinant viruses were engineered to express the glycoprotein (G) of avian metapneumovirus (aMPV), subtype A, B or C, as bivalent vaccines. These recombinant viruses were slightly attenuated in vivo, yet maintaine...

  9. Enhancement of feline immunodeficiency virus infection after immunization with envelope glycoprotein subunit vaccines.

    NARCIS (Netherlands)

    C.H.J. Siebelink (Kees); E.J. Tijhaar (Edwin); R.C. Huisman (Robin); W. Huisman (Willem); A. de Ronde; I.H. Darby; M.J. Francis; G.F. Rimmelzwaan (Guus); A.D.M.E. Osterhaus (Albert)

    1995-01-01

    textabstractCats were immunized three times with different recombinant feline immunodeficiency virus (FIV) candidate vaccines. Recombinant vaccinia virus (rVV)-expressed envelope glycoprotein with (vGR657) or without (vGR657 x 15) the cleavage site and an FIV envelope bacterial fusion protein

  10. Improved method for silver staining of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels

    DEFF Research Database (Denmark)

    Møller, H J; Poulsen, J H

    1995-01-01

    A method for detection of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels was developed by a combination of (i) initial periodic acid oxidation/Alcian blue staining and (ii) subsequent staining with silver nitrate. The procedure allowed detection of as little as 1.6 ng of alpha 1...

  11. Interactions between P-glycoprotein substrates and other cationic drugs at the hepatic excretory level

    NARCIS (Netherlands)

    Smit, JW; Duin, E; Steen, H; Roggeveld, J; Meijer, DKF

    1 In the present study it was tested whether known P-glycoprotein (P-gp) substrates/MDR reversal agents interact with small (type 1) and bulky (type 2) cationic drugs at the level of biliary excretion in the rat isolated perfused liver model (IPRL). The studies were performed with model compounds

  12. Molecular characterization and baculovirus expression of the glycoprotein B of a seal herpesvirus (phocid herpesvirus-1).

    NARCIS (Netherlands)

    T.C. Harder (Timm); A.D.M.E. Osterhaus (Albert)

    1997-01-01

    textabstractA glycoprotein B (gB) gene homologue was identified in a 5.4-kb BamHl genomic fragment of the phocid herpesvirus type-1 (PhHV-1) which represents a widespread and important pathogen of pinnipeds. Sequence analysis revealed a gB-specific open-reading frame comprising 881 amino acids.

  13. Screening for the P-Glycoprotein Inhibitory Pump Activity of Plant ...

    African Journals Online (AJOL)

    6G as the fluorescent probe and reserpine, a known inhibitor of P-glycoprotein pump, was used as a reference drug. The results revealed that out of the 45 plant extracts tested, 3 .... line and it was first obtained from the pleural effkion of a female cancer patient. MCF-7 resistant (MCF-7R) cells may be obtained by incubating ...

  14. Cytomegalovirus glycoprotein B genotyping in ocular fluids and blood of AIDS patients with cytomegalovirus retinitis

    NARCIS (Netherlands)

    Peek, R.; Verbraak, F.; Bruinenberg, M.; van der Lelij, A.; van den Horn, G.; Kijlstra, A.

    1998-01-01

    To determine the frequency of cytomegalovirus glycoprotein B (gB) genotypes in clinical samples of ocular fluids of patients with acquired immune deficiency syndrome (AIDS) who have cytomegalovirus retinitis and to compare these with the cytomegalovirus gB genotype in paired peripheral blood

  15. Variations in Spike Glycoprotein Gene of MERS-CoV, South Korea, 2015.

    Science.gov (United States)

    Kim, Dae-Won; Kim, You-Jin; Park, Sung Han; Yun, Mi-Ran; Yang, Jeong-Sun; Kang, Hae Ji; Han, Young Woo; Lee, Han Saem; Kim, Heui Man; Kim, Hak; Kim, A-Reum; Heo, Deok Rim; Kim, Su Jin; Jeon, Jun Ho; Park, Deokbum; Kim, Joo Ae; Cheong, Hyang-Min; Nam, Jeong-Gu; Kim, Kisoon; Kim, Sung Soon

    2016-01-01

    An outbreak of nosocomial infections with Middle East respiratory syndrome coronavirus occurred in South Korea in May 2015. Spike glycoprotein genes of virus strains from South Korea were closely related to those of strains from Riyadh, Saudi Arabia. However, virus strains from South Korea showed strain-specific variations.

  16. Immunoinformatic Analysis of Crimean Congo Hemorrhagic Fever Virus Glycoproteins and Epitope Prediction for Synthetic Peptide Vaccine

    International Nuclear Information System (INIS)

    Tipu, H. N.

    2016-01-01

    Objective: To determine the Crimean Congo Hemorrhagic Fever (CCHF) virus M segement glycoprotein's immunoinformatic parameters, and identify Human Leukocyte Antigen (HLA) class I binders as candidates for synthetic peptide vaccines. Study Design: Cross-sectional study. Place and Duration of Study: Combined Military Hospital, Khuzdar Cantt, in May 2015. Methodology: Data acquisition, antigenicity prediction, secondary and tertiary structure prediction, residue analysis were done using immunoinformatics tools. HLA class I binders in glycoprotein's sequence were identified at nanomer length using NetMHC 3.4 and mapped onto tertiary structure. Docking was done for strongest binder against its corresponding allele with CABS-dock. Results: HLA A*0101, 0201, 0301, 2402, 2601 and B*0702, 0801, 2705, 3901, 4001, 5801, 1501 were analyzed against two glycoprotein components of the virus. A total of 35 nanomers from GP1, and 3 from GP2 were identified. HLA B*0702 bound maximum number of peptides (6), while HLA B*4001 showed strongest binding affinity. Conclusion: HLA specific glycoproteins epitope prediction can help identify synthetic peptide vaccine candidates. (author)

  17. Virulence determinants within the E2 glycoprotein of Classical Swine Fever Virus

    DEFF Research Database (Denmark)

    Johnston, Camille Melissa; Fahnøe, Ulrik; Lohse, Louise

    Classical Swine Fever is a highly contagious disease of pigs caused by Classical Swine Fever Virus (CSFV), a member of the pestivirus genus within the family Flaviviridae. The E2 glycoprotein of CSFV has been shown to be an important factor for the virulence of the virus. In a recent study, we have...

  18. Structure of a Pestivirus Envelope Glycoprotein E2 Clarifies Its Role in Cell Entry

    Directory of Open Access Journals (Sweden)

    Kamel El Omari

    2013-01-01

    Full Text Available Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1 at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed.

  19. Structure of a Pestivirus Envelope Glycoprotein E2 Clarifies Its Role in Cell Entry

    Science.gov (United States)

    El Omari, Kamel; Iourin, Oleg; Harlos, Karl; Grimes, Jonathan M.; Stuart, David I.

    2013-01-01

    Summary Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed. PMID:23273918

  20. Characterization of Vesicular Stomatitis Virus Pseudotypes Bearing Essential Entry Glycoproteins gB, gD, gH, and gL of Herpes Simplex Virus 1.

    Science.gov (United States)

    Rogalin, Henry B; Heldwein, Ekaterina E

    2016-11-15

    Herpes simplex viruses (HSVs) are unusual in that unlike most enveloped viruses, they require at least four entry glycoproteins, gB, gD, gH, and gL, for entry into target cells in addition to a cellular receptor for gD. The dissection of the herpes simplex virus 1 (HSV-1) entry mechanism is complicated by the presence of more than a dozen proteins on the viral envelope. To investigate HSV-1 entry requirements in a simplified system, we generated vesicular stomatitis virus (VSV) virions pseudotyped with HSV-1 essential entry glycoproteins gB, gD, gH, and gL but lacking the native VSV fusogen G. These virions, referred to here as VSVΔG-BHLD virions, infected a cell line expressing a gD receptor, demonstrating for the first time that the four essential entry glycoproteins of HSV-1 are not only required but also sufficient for cell entry. To our knowledge, this is the first time the VSV pseudotyping system has been successfully extended beyond two proteins. Entry of pseudotyped virions required a gD receptor and was inhibited by HSV-1 specific anti-gB or anti-gH/gL neutralizing antibodies, which suggests that membrane fusion during the entry of the pseudotyped virions shares common requirements with the membrane fusion involved in HSV-1 entry and HSV-1-mediated syncytium formation. The HSV pseudotyping system established in this study presents a novel tool for systematic exploration of the HSV entry and membrane fusion mechanisms. Herpes simplex viruses (HSVs) are human pathogens that can cause cold sores, genital herpes, and blindness. No vaccines or preventatives are available. HSV entry into cells-a prerequisite for a successful infection-is a complex process that involves multiple viral and host proteins and occurs by different routes. Detailed mechanistic knowledge of the HSV entry is important for understanding its pathogenesis and would benefit antiviral and vaccine development, yet the presence of more than a dozen proteins on the viral envelope complicates

  1. Comparison of α1-Antitrypsin, α1-Acid Glycoprotein, Fibrinogen and NOx as Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis).

    Science.gov (United States)

    Guha, Anirban; Guha, Ruby; Gera, Sandeep

    2013-06-01

    Mastitis set apart as clinical and sub clinical is a disease complex of dairy cattle, with sub clinical being the most important economically. Of late, laboratories showed interest in developing biochemical markers to diagnose sub clinical mastitis (SCM) in herds. Many workers reported noteworthy alternation of acute phase proteins (APPs) and nitric oxide, (measured as nitrate+nitrite = NOx) in milk due to intra-mammary inflammation. But, the literature on validation of these parameters as indicators of SCM, particularly in riverine milch buffalo (Bubalus bubalis) milk is inadequate. Hence, the present study focused on comparing several APPs viz. α1- anti trypsin, α1- acid glycoprotein, fibrinogen and NOx as indicators of SCM in buffalo milk. These components in milk were estimated using standardized analytical protocols. Somatic cell count (SCC) was done microscopically. Microbial culture was done on 5% ovine blood agar. Of the 776 buffaloes (3,096 quarters) sampled, only 347 buffaloes comprising 496 quarters were found positive for SCM i.e. milk culture showed growth in blood agar with SCC≥2×10(5) cells/ml of milk. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. It was observed that α1- anti trypsin and NOx had a highly significant (pSCM milk, whereas, the increase of α1- acid glycoprotein in infected milk was significant (pSCM milk. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×10(5) cells/ml of milk as the benchmark. Udder profile correlation coefficient was also used. Allowing for statistical and epidemiological analysis, it was concluded that α1- anti trypsin indicates SCM irrespective of etiology, whereas α1- acid glycoprotein better diagnosed SCM caused by gram positive bacteria. NOx did not prove to be a good indicator of SCM. It is recommended measuring both α1- anti trypsin and α1

  2. Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation.

    Science.gov (United States)

    Ghaderi, Darius; Zhang, Mai; Hurtado-Ziola, Nancy; Varki, Ajit

    2012-01-01

    One of the fastest growing fields in the pharmaceutical industry is the market for therapeutic glycoproteins. Today, these molecules play a major role in the treatment of various diseases, and include several protein classes, i.e., clotting factors, hormones, cytokines, antisera, enzymes, enzyme inhibitors, Ig-Fc-Fusion proteins, and monoclonal antibodies. Optimal glycosylation is critical for therapeutic glycoproteins, as glycans can influence their yield, immunogenicity and efficacy, which impact the costs and success of such treatments. While several mammalian cell expression systems currently used can produce therapeutic glycoproteins that are mostly decorated with human-like glycans, they can differ from human glycans by presenting two structures at the terminal and therefore most exposed position. First, natural human N-glycans are lacking the terminal Gal 1-3Gal (alpha-Gal) modification; and second, they do not contain the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). All humans spontaneously express antibodies against both of these glycan structures, risking increased immunogenicity of biotherapeutics carrying such non-human glycan epitopes. However, in striking contrast to the alpha-Gal epitope, exogenous Neu5Gc can be metabolically incorporated into human cells and presented on expressed glycoproteins in several possible epitopes. Recent work has demonstrated that this non-human sialic acid is found in widely varying amounts on biotherapeutic glycoproteins approved for treatment of various medical conditions. Neu5Gc on glycans of these medical agents likely originates from the production process involving the non-human mammalian cell lines and/or the addition of animal-derived tissue culture supplements. Further studies are needed to fully understand the impact of Neu5Gc in biotherapeutic agents. Similar concerns apply to human cells prepared for allo- or auto-transplantation, that have been grown in animal-derived tissue culture supplements.

  3. MoFi: A Software Tool for Annotating Glycoprotein Mass Spectra by Integrating Hybrid Data from the Intact Protein and Glycopeptide Level.

    Science.gov (United States)

    Skala, Wolfgang; Wohlschlager, Therese; Senn, Stefan; Huber, Gabriel E; Huber, Christian G

    2018-04-18

    Hybrid mass spectrometry (MS) is an emerging technique for characterizing glycoproteins, which typically display pronounced microheterogeneity. Since hybrid MS combines information from different experimental levels, it crucially depends on computational methods. Here, we describe a novel software tool, MoFi, which integrates hybrid MS data to assign glycans and other post-translational modifications (PTMs) in deconvoluted mass spectra of intact proteins. Its two-stage search algorithm first assigns monosaccharide/PTM compositions to each peak and then compiles a hierarchical list of glycan combinations compatible with these compositions. Importantly, the program only includes those combinations which are supported by a glycan library as derived from glycopeptide or released glycan analysis. By applying MoFi to mass spectra of rituximab, ado-trastuzumab emtansine, and recombinant human erythropoietin, we demonstrate how integration of bottom-up data may be used to refine information collected at the intact protein level. Accordingly, our software reveals that a single mass frequently can be explained by a considerable number of glycoforms. Yet, it simultaneously ranks proteoforms according to their probability, based on a score which is calculated from relative glycan abundances. Notably, glycoforms that comprise identical glycans may nevertheless differ in score if those glycans occupy different sites. Hence, MoFi exposes different layers of complexity that are present in the annotation of a glycoprotein mass spectrum.

  4. Glycoprotein isolated from Ulmus davidiana NAKAI protects against carbon tetrachloride-induced liver injury in the mouse.

    Science.gov (United States)

    Ko, Jeong-Hyeon; Lim, Kye-Taek

    2006-07-01

    Ulmus davidiana NAKAI (UDN) has traditionally been used for healing of inflammatory diseases. This study was carried out to investigate the hepatoprotective effect of the glycoprotein isolated from UDN in carbon tetrachloride (CCl4)-induced liver injury. We evaluated the activities of alanine aminotransferase (ALT), lactate dehydrogenase (LDH), thiobarbituric acid-reactive substances (TBARS), and antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)] activities in CCl4-treated mice. When mice were treated with CCl4 in the absence of UDN glycoprotein, the activities of ALT, LDH, and TBARS were increased, while the antioxidant enzymes activities were decreased. However, when the mice were treated with CCl4 in the presence of UDN glycoprotein, the activities of ALT, LDH, and TBARS were significantly reduced and SOD, CAT, and GPx activities were remarkably increased. In addition, UDN glycoprotein increased the nitric oxide production and decreased the nuclear factor-kappa B and activator protein-1 activation in CCl4-treated mice. We also investigated the protective effects of UDN glycoprotein in glucose/glucose oxidase (G/GO)-induced cytotoxicity in primary cultured mouse hepatocytes. UDN glycoprotein markedly inhibited the cell death induced by G/GO. These results suggest that UDN glycoprotein protects against CCl4-induced liver injury in the mouse.

  5. Glycoprotein isolated from Ulmus davidiana Nakai regulates expression of iNOS and COX-2 in vivo and in vitro.

    Science.gov (United States)

    Lee, Sei-Jung; Lim, Kye-Taek

    2007-06-01

    This study was carried out to investigate the anti-inflammatory potential of a 116-kDa glycoprotein isolated from Ulmus davidiana Nakai (UDN glycoprotein, 116 kDa) in lipopolysaccaride (LPS)-treated RAW 264.7 cells and dextran sodium sulfate (DSS)-treated A/J mouse. In LPS (1 microg/ml)-stimulated RAW 264.7 cells, we found that UDN glycoprotein has dose-dependent blocking effects of reactive oxygen species (ROS) and inducible nitric oxide (NO) production. In addition, the results obtained from electrophoretic mobility shift assay (EMSA) and western blot analysis showed that UDN glycoprotein dose-dependently inhibits DNA binding activity of nuclear factor-kappa B (NF-kappaB), and activities of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and manganese-superoxide dismutases (Mn-SOD) in LPS-stimulated RAW 264.7 cells. Similar results after treatment with UDN glycoprotein were also brought in the DSS-stimulated A/J mouse colitis. The increased disease activity index (DAI) and the shortened large intestine in DSS (5%)-treated A/J mouse were normalized by treatment with UDN glycoprotein [40 mg/kg body weight (BW)]. These intestinal protective activities of UDN glycoprotein are caused by blockage of plasmic thiobarbituric acid reactive substances (TBARS) formation, nitric oxide (NO) production, and lactate dehydrogenase (LDH) release, accompanying the inhibition of colonic inflammatory signal mediators (NF-kappaB, iNOS, and COX-2). These results in this study were presumably come from anti-oxidative effect of UDN glycoprotein in either LPS-stimulated RAW 264.7 cells or DSS-stimulated A/J mouse colitis. Therefore, we speculate that UDN glycoprotein has anti-inflammatory potential at the early inflammation stage.

  6. An immune stimulating complex (iscom) subunit rabies vaccine protects dogs and mice against street rabies challenge.

    NARCIS (Netherlands)

    M. Fekadu; J.H. Schaddock; J. Ekströ m; A.D.M.E. Osterhaus (Albert); D.W. Sanderlin; B. Sundquist; B. Morein (Bror)

    1992-01-01

    textabstractDogs and mice were immunized with either a rabies glycoprotein subunit vaccine incorporated into an immune stimulating complex (ISCOM) or a commercial human diploid cell vaccine (HDCV) prepared from a Pitman Moore (PM) rabies vaccine strain. Pre-exposure vaccination of mice with two

  7. Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    S Gnanakaran

    Full Text Available A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an

  8. Conglutinin binds the HIV-1 envelope glycoprotein gp 160 and inhibits its interaction with cell membrane CD4

    DEFF Research Database (Denmark)

    Andersen, Ove; Sørensen, A M; Svehag, S E

    1991-01-01

    The highly glycosylated envelope glycoprotein (gp 160) of human immunodeficiency virus (HIV) interacts with the CD4 molecule present on the membrane of CD4+ cells and is involved in the pathobiology of HIV infection. Lectins bind glycoproteins through non-covalent interactions with specific hexose...... of the binding of rgp160 to the CD4 receptor on CEM 13 cells, as demonstrated by FACS analyses. These results indicate that conglutinin may inhibit the infection with HIV-1 through its interaction with the viral envelope glycoprotein....

  9. Elicitation of neutralizing antibodies directed against CD4-induced epitope(s using a CD4 mimetic cross-linked to a HIV-1 envelope glycoprotein.

    Directory of Open Access Journals (Sweden)

    Antu K Dey

    Full Text Available The identification of HIV-1 envelope glycoprotein (Env structures that can generate broadly neutralizing antibodies (BNAbs is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4 receptor-bound state, thereby exposing highly conserved "CD4 induced" (CD4i epitope(s known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH, was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140 using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1 complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-2(7312/V434M and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s. These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s here, and its potential role in vaccine application.

  10. Evaluation of ion mobility for the separation of glycoconjugate isomers due to different types of sialic acid linkage, at the intact glycoprotein, glycopeptide and glycan level.

    Science.gov (United States)

    Barroso, Albert; Giménez, Estela; Konijnenberg, Albert; Sancho, Jaime; Sanz-Nebot, Victoria; Sobott, Frank

    2018-02-20

    The study of protein glycosylation can be regarded as an intricate but very important task, making glycomics one of the most challenging and interesting, albeit under-researched, type of "omics" science. Complexity escalates remarkably when considering that carbohydrates can form severely branched structures with many different constituents, which often leads to the formation of multiple isomers. In this regard, ion mobility (IM) spectrometry has recently demonstrated its power for the separation of isomeric compounds. In the present work, the potential of traveling wave IM (TWIMS) for the separation of isomeric glycoconjugates was evaluated, using mouse transferrin (mTf) as model glycoprotein. Particularly, we aim to assess the performance of this platform for the separation of isomeric glycoconjugates due to the type of sialic acid linkage, at the intact glycoprotein, glycopeptide and glycan level. Straightforward separation of isomers was achieved with the analysis of released glycans, as opposed to the glycopeptides which showed a more complex pattern. Finally, the developed methodology was applied to serum samples of mice, to investigate its robustness when analyzing real complex samples. Ion mobility mass spectrometry is a promising analytical technique for the separation of glycoconjugate isomers due to type of sialic acid linkage. The impact of such a small modification in the glycan structure is more evident in smaller analytes, reason why the analysis of free glycans was easier compared to the intact protein or the glycopeptides. The established methodology could be regarded as starting point in the separation of highly decorated glycoconjugates. This is an important topic nowadays, as differences in the abundance of some glycan isomers could be the key for the early diagnosis, control or differentiation of certain diseases, such as inflammation or cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Activation and Inactivation of Primary Human Immunodeficiency Virus Envelope Glycoprotein Trimers by CD4-Mimetic Compounds

    Science.gov (United States)

    Madani, Navid; Princiotto, Amy M.; Zhao, Connie; Jahanbakhshsefidi, Fatemeh; Mertens, Max; Herschhorn, Alon; Melillo, Bruno; Smith, Amos B.

    2016-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the viral envelope glycoproteins (Env), a trimer of three gp120 exterior glycoproteins, and three gp41 transmembrane glycoproteins. The metastable Env is triggered to undergo entry-related conformational changes when gp120 binds sequentially to the receptors, CD4 and CCR5, on the target cell. Small-molecule CD4-mimetic compounds (CD4mc) bind gp120 and act as competitive inhibitors of gp120-CD4 engagement. Some CD4mc have been shown to trigger Env prematurely, initially activating Env function, followed by rapid and irreversible inactivation. Here, we study CD4mc with a wide range of anti-HIV-1 potencies and demonstrate that all tested CD4mc are capable of activating as well as inactivating Env function. Biphasic dose-response curves indicated that the occupancy of the protomers in the Env trimer governs viral activation versus inactivation. One CD4mc bound per Env trimer activated HIV-1 infection. Envs with two CD4mc bound were activated for infection of CD4-negative, CCR5-positive cells, but the infection of CD4-positive, CCR5-positive cells was inhibited. Virus was inactivated when all three Env protomers were occupied by the CD4mc, and gp120 shedding from the Env trimer was increased in the presence of some CD4mc. Env reactivity and the on rates of CD4mc binding to the Env trimer were found to be important determinants of the potency of activation and entry inhibition. Cross-sensitization of Env protomers that do not bind the CD4mc to neutralization by an anti-V3 antibody was not evident. These insights into the mechanism of antiviral activity of CD4mc should assist efforts to optimize their potency and utility. IMPORTANCE The trimeric envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) mediate virus entry into host cells. Binding to the host cell receptors, CD4 and CCR5, triggers changes in the conformation of the HIV-1 envelope glycoprotein trimer important

  12. Rhodocytin (aggretin) activates platelets lacking alpha(2)beta(1) integrin, glycoprotein VI, and the ligand-binding domain of glycoprotein Ibalpha

    DEFF Research Database (Denmark)

    Bergmeier, W; Bouvard, D; Eble, J A

    2001-01-01

    Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from...... the venom of Calloselasma rhodostoma, induces platelet activation that can be blocked by monoclonal antibodies against alpha(2)beta(1) integrin. This finding suggested that clustering of alpha(2)beta(1) integrin by rhodocytin is sufficient to induce platelet activation and led to the hypothesis...

  13. P-glycoprotein epitope mapping. II. The murine monoclonal antibody MM6.15 to human multidrug-resistant cells binds with three distinct loops in the MDR1-P-glycoprotein extracellular domain.

    Science.gov (United States)

    Cianfriglia, M; Romagnoli, G; Tombesi, M; Poloni, F; Falasca, G; Di Modugno, F; Castagna, M; Chersi, A

    1995-03-29

    A new murine monoclonal antibody (MAb), MM6.15, to human MDR1 P-glycoprotein was found to be reactive in ELISA with synthetic peptides selected from the predicted sequences of the first, fourth and sixth extracellular loop of MDR1-P-glycoprotein. In order to precisely define the MM6.15-binding site, a peptide library of overlapping 5- to 9-mer residues covering the entire sixth extracellular loop of both human and rodent class-1 P-glycoproteins was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MAb MM6.15 reacts only with human synthetic peptides and that the critical component of the MAb recognition is made up of the amino-acid sequence LVAHKL (residues 963-968 of the MDR1-P-glycoprotein) with histidine (H), lysine (K) and possibly leucine (L), key residues of this immunogenic domain.

  14. Sestamibi is a substrate for MDR1 and MDR2 P-glycoprotein genes

    Energy Technology Data Exchange (ETDEWEB)

    Joseph, Brigid; Malhi, Harmeet; Gupta, Sanjeev [Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Ullmann 625, 1300 Morris Park Avenue, NY 10461, Bronx (United States); Bhargava, Kuldeep K.; Palestro, Christopher J. [Division of Nuclear Medicine, Long Island Jewish Medical Center, New York (United States); Schilsky, Michael L. [Division of Liver Diseases, Mount Sinai School of Medicine, New York (United States); Jain, Diwakar [Division of Nuclear Cardiology, MCP-Hahnemann University School of Medicine, Philadephia (United States)

    2003-07-01

    Technetium-99m sestamibi has attracted interest for assessment of the function of P-glycoproteins, which are well expressed in the liver and have roles in biliary transport and the removal of chemotherapeutic drugs. To further examine the cross-reactivity of {sup 99m}Tc-sestamibi for P-glycoprotein family members, we conducted studies in animals. Hepatobiliary secretion of {sup 99m}Tc-sestamibi was determined in normal FVB/N mice, mutant mice with specific P-glycoprotein deficiencies in the FVB/N background, normal Long-Evans Agouti (LEA) rats, and Long-Evans Cinnamon (LEC) rats with abnormal copper transport and liver disease but intact P-glycoprotein expression. After intrasplenic injection, {sup 99m}Tc-sestamibi was rapidly incorporated in the mouse and rat liver, with maximal accumulation after 102{+-}31 and 109{+-}16 s, respectively (P=NS). In normal mice and rats, 55%{+-}11% and 55%{+-}6%, respectively, of the maximal sestamibi activity was retained in the liver after 1 h (P=NS). In double knockout mice lacking both mdr1a and mdr1b homologs of the human MDR1 (ABCB1) gene, 88%{+-}11% of maximal sestamibi activity was retained in the liver after 1 h (P<0.001). In knockout mice deficient in either mdr1a gene or mdr2 (ABCB4) gene, biliary sestamibi excretion was also impaired, although this impairment was relatively less pronounced in ABCB4-deficient mice than in double knockout mice lacking both ABCB1 gene homologs (P<0.03). Hepatobiliary sestamibi excretion in LEC rats was not different from that in control normal rats, despite the presence of significant liver disease in the former. Hepatobiliary sestamibi excretion requires P-glycoproteins and is unperturbed in chronic liver disease. Sestamibi appears to be a substrate for both ABCB1 and ABCB4 genes, although the former utilizes it far more efficiently. Assessment of P-glycoprotein activity with sestamibi should consider how regulation of ABCB1 and related family members might modulate sestamibi incorporation

  15. A Critical View on In Vitro Analysis of P-glycoprotein (P-gp) Transport Kinetics.

    Science.gov (United States)

    Saaby, Lasse; Brodin, Birger

    2017-09-01

    Transport proteins expressed in the different barriers of the human body can have great implications on absorption, distribution, and excretion of drug compounds. Inhibition or saturation of a transporter can potentially alter these absorbtion, distribution, metabolism and elimination properties and thereby also the pharmacokinetic profile and bioavailability of drug compounds. P-glycoprotein (P-gp, ABCB1) is an efflux transporter which is present in most of the barriers of the body, including the small intestine, the blood-brain barrier, the liver, and the kidney. In all these tissues, P-gp may mediate efflux of drug compounds and may also be a potential site for drug-drug interactions. Consequently, there is a need to be able to predict the saturation and inhibition of P-gp and other transporters in vivo. For this purpose, Michaelis-Menten steady-state analysis has been applied to estimate kinetic parameters, such as K m and V max , for carrier-mediated transport, whereas half-maximal inhibitor concentration (IC 50 ) and the disassociation constant for an inhibitor/P-gp complex (K i ) have been determined to estimate P-gp inhibition. This review addresses in vitro methods commonly used to study P-gp transport kinetics and aims at providing a critical evaluation of the application of steady-state Michaelis-Menten analysis of kinetic parameters for substrate/P-gp interactions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  16. Functional Regulation of the Plasma Protein Histidine-Rich Glycoprotein by Zn2+ in Settings of Tissue Injury

    Directory of Open Access Journals (Sweden)

    Kristin M. Priebatsch

    2017-03-01

    Full Text Available Divalent metal ions are essential nutrients for all living organisms and are commonly protein-bound where they perform important roles in protein structure and function. This regulatory control from metals is observed in the relatively abundant plasma protein histidine-rich glycoprotein (HRG, which displays preferential binding to the second most abundant transition element in human systems, Zinc (Zn2+. HRG has been proposed to interact with a large number of protein ligands and has been implicated in the regulation of various physiological and pathological processes including the formation of immune complexes, apoptotic/necrotic and pathogen clearance, cell adhesion, antimicrobial activity, angiogenesis, coagulation and fibrinolysis. Interestingly, these processes are often associated with sites of tissue injury or tumour growth, where the concentration and distribution of Zn2+ is known to vary. Changes in Zn2+ levels have been shown to modify HRG function by altering its affinity for certain ligands and/or providing protection against proteolytic disassembly by serine proteases. This review focuses on the molecular interplay between HRG and Zn2+, and how Zn2+ binding modifies HRG-ligand interactions to regulate function in different settings of tissue injury.

  17. Antigenicity of peptides comprising the immunosuppressive domain of the retroviral envelope glycoprotein [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Bryony Jenkins

    2016-12-01

    Full Text Available To achieve persistent infection of the host, viruses often subvert or suppress host immunity through mechanisms that are not entirely understood. The envelope glycoprotein of several retroviruses is thought to possess potent immunosuppressive activity, mapped to a 17-amino acid residue conserved domain. Synthetic peptides corresponding to this immunosuppressive domain can inhibit lymphocyte activation, whereas mutation of key domain residues can increase the lymphocyte response to linked antigenic epitopes. Using three T cell receptors (TCRs of defined specificity, we examine the effect of the immunosuppressive domain on the T cell response to their respective antigenic peptides. We find that fusion of a T cell epitope to the immunosuppressive domain can greatly modulate its potency. However, the effects heavily depend on the particular combination of TCR and peptide-major histocompatibility complex class II (pMHC II, and are mimicked by sequence-scrambled peptides of similar length, suggesting they operate at the level of TCR-pMHC interaction. These results offer an alternative explanation for the immunogenicity of T cell epitopes comprising the putative immunosuppressive domain, which is more consistent with an effect on peptide antigenicity than true immunosuppressive activity.

  18. Antigenicity of peptides comprising the immunosuppressive domain of the retroviral envelope glycoprotein [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Bryony Jenkins

    2017-02-01

    Full Text Available To achieve persistent infection of the host, viruses often subvert or suppress host immunity through mechanisms that are not entirely understood. The envelope glycoprotein of several retroviruses is thought to possess potent immunosuppressive activity, mapped to a 17-amino acid residue conserved domain. Synthetic peptides corresponding to this immunosuppressive domain can inhibit lymphocyte activation, whereas mutation of key domain residues can increase the lymphocyte response to linked antigenic epitopes. Using three T cell receptors (TCRs of defined specificity, we examine the effect of the immunosuppressive domain on the T cell response to their respective antigenic peptides. We find that fusion of a T cell epitope to the immunosuppressive domain can greatly modulate its potency. However, the effects heavily depend on the particular combination of TCR and peptide-major histocompatibility complex class II (pMHC II, and are mimicked by sequence-scrambled peptides of similar length, suggesting they operate at the level of pMHC formation or TCR-pMHC interaction. These results offer an alternative explanation for the immunogenicity of T cell epitopes comprising the putative immunosuppressive domain, which is more consistent with an effect on peptide antigenicity than true immunosuppressive activity.

  19. Structure of Epstein-Barr Virus Glycoprotein 42 Suggests a Mechanism for Triggering Receptor-Activated Virus Entry

    Energy Technology Data Exchange (ETDEWEB)

    Kirschner, Austin N.; Sorem, Jessica; Longnecker, Richard; Jardetzky, Theodore S.; (NWU); (Stanford-MED)

    2009-05-26

    Epstein-Barr virus requires glycoproteins gH/gL, gB, and gp42 to fuse its lipid envelope with B cells. Gp42 is a type II membrane protein consisting of a flexible N-terminal region, which binds gH/gL, and a C-terminal lectin-like domain that binds to the B-cell entry receptor human leukocyte antigen (HLA) class II. Gp42 triggers membrane fusion after HLA binding, a process that requires simultaneous binding to gH/gL and a functional hydrophobic pocket in the lectin domain adjacent to the HLA binding site. Here we present the structure of gp42 in its unbound form. Comparisons to the previously determined structure of a gp42:HLA complex reveals additional N-terminal residues forming part of the gH/gL binding site and structural changes in the receptor binding domain. Although the core of the lectin domain remains similar, significant shifts in two loops and an {alpha} helix bordering the essential hydrophobic pocket suggest a structural mechanism for triggering fusion.

  20. Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection.

    Science.gov (United States)

    Oliver, Stefan L; Yang, Edward; Arvin, Ann M

    2017-01-01

    The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles. Postherpetic

  1. Mice orally immunized with a transgenic plant expressing the glycoprotein of Crimean-Congo hemorrhagic fever virus

    DEFF Research Database (Denmark)

    Ghiasi, Seyed Mojtaba; Salmanian, A H; Chinikar, S

    2011-01-01

    glycoprotein when expressed in the root and leaf of transgenic plants via hairy roots and stable transformation of tobacco plants, respectively. After confirmatory analyses of transgenic plant lines and quantification of the expressed glycoprotein, mice were either fed with the transgenic leaves or roots, fed...... the transgenic plant material and injected subcutaneously with the plant-made CCHFV glycoprotein (fed/boosted), vaccinated with an attenuated CCHF vaccine (positive control), or received no treatment (negative control). All immunized groups had a consistent rise in anti-glycoprotein IgG and IgA antibodies...... in their serum and feces, respectively. The mice in the fed/boosted group showed a significant rise in specific IgG antibodies after a single boost. Our results imply that oral immunization of animals with edible materials from transgenic plants is feasible, and further assessments are under way. In addition...

  2. Hydroxyproline-rich glycoproteins accumulate in pearl millet after seed treatment with elicitors of defense responses against Sclerospora graminicola

    NARCIS (Netherlands)

    Sujeeth, Neerakkal; Deepak, Shantharaj; Shailasree, Sekhar; Kini, Ramachandra K.; Shetty, Shekar H.; Hille, Jacques

    The accumulation of hydroxyproline-rich glycoproteins (HRGPs) was investigated after induction of resistance in pearl millet against downy mildew caused by Sclerospora graminicola. Treatment of susceptible pearl millet seeds with various biotic and abiotic elicitors resulted in increased HRGP

  3. P-glycoprotein epitope mapping. I. Identification of a linear human-specific epitope in the fourth loop of the P-glycoprotein extracellular domain by MM4.17 murine monoclonal antibody to human multi-drug-resistant cells.

    Science.gov (United States)

    Cianfriglia, M; Willingham, M C; Tombesi, M; Scagliotti, G V; Frasca, G; Chersi, A

    1994-01-02

    A new murine monoclonal antibody (MAb), MM4.17, to human multi-drug-resistant (MDR) cells was found to be reactive in an ELISA with a synthetic 16-amino acid peptide selected from the fourth loop of the P-glycoprotein extracellular domain. Immunohistochemistry indicated that this MAb reacted in human tissues in the same pattern as that previously found with other human-specific MAbs to P-glycoprotein. For a precise definition of the MM4.17 epitope, a peptide library consisting of overlapping 4- to 10-mer residues covering the entire P-glycoprotein-fragment was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MM4.17 epitope is constituted by the continuous-linear TRIDDPET amino-acid sequence (residues 750-757 of the human MDRI-P-glycoprotein). The MAb MM4.17 recognizes only the human MDRI-P-glycoprotein isoform, and excess TRIDDPET peptide blocks the binding of the MAb to MDR variants of CEM cells. These results demonstrate that the amino-acid sequence TRIDDPET from the human MDRI gene represents the first continuous-linear epitope identified in the P-glycoprotein extracellular domain.

  4. Análise morfológica, imunofenotípica e molecular na identificação da leucemia megacariocítica aguda (LMA-M7 Morphologic, cytogenetic and molecular analyses in the identification of acute megakaryocytic leukemia (AML-M7

    Directory of Open Access Journals (Sweden)

    Mariela G. Farias

    2007-12-01

    represents from 3 to 5% of AML cases, is frequently associated to myelofibrosis and is a subtype with poor prognosis. The diagnosis of AML-M7 is initially based on the morphologic characteristics of leukemic cells. Bone marrow aspirates or biopsy shows a population of pleomorphic and basophilic cells which may present cytoplasmatic blebs. Morphologic and cytochemical criteria are not enough for a correct diagnosis. It is necessary to differentiate acute megakaryocytic leukemia with other subtypes of acute myeloid leukemia mainly in cases in which blast cells are undifferentiated, as are the cases of AML-M0, AML-M1 and of acute lymphoid leukemia, subtypes L1 and L2. The use of immunopherotypic techniques is essential for a differential diagnosis showing a population of leukemia cells with the absence of most surface lymphoid and myeloid markers, yet relevant for the antigen of the megakaryocytic expression: CD41a (complex glycoproteic IIb/IIIa, CD42b (glycoprotein Ib and/or CD61 (glycoprotein IIIa, there by providing the for correct classification in 98% of the cases. Chromosomal abnormalities identified by cytogenetic techniques and molecular analysis are important for determining the prognosis and definition of the therapheutic regime.

  5. Partial Characterization of a Vicilin-Like Glycoprotein from Seeds of Flowering Tobacco (Nicotiana sylvestris

    Directory of Open Access Journals (Sweden)

    Jared Q. Gerlach

    2009-01-01

    Full Text Available A vicilin-like glycoprotein from the seeds of Nicotiana sylvestris, flowering tobacco, has been identified using nanoLC/ESI-MS/MS. Sequences from a fragment of protein demonstrated homology with vicilins from other members of the Solanaceae family, notably potato (Solanum demissum. Reducing and nonreducing SDS-PAGE analyses of the identified protein indicated that fragments resulting from in situ proteolytic processing are joined by intrachain disulphide bonds. Staining with Con A lectin was specifically inhibited by mannose suggested the presence of -linked glycosylation which was confirmed by carbohydrate compositional analysis of PVDF-bound protein subunits. HPAEC-PAD analysis of the monosaccharides released from the glycoprotein by acid hydrolysis revealed glucosamine and mannose. -acetylglucosamine termination of attached oligosaccharides was further verified by inhibitable WGA lectin staining. Immunostaining of PVDF-bound N. sylvestris proteins with antibodies against G. max total protein demonstrated cross-staining at masses corresponding to fragments from the proteolytically processed protein subunits.

  6. Defining the antibody cross-reactome directed against the influenza virus surface glycoproteins.

    Science.gov (United States)

    Nachbagauer, Raffael; Choi, Angela; Hirsh, Ariana; Margine, Irina; Iida, Sayaka; Barrera, Aldo; Ferres, Marcela; Albrecht, Randy A; García-Sastre, Adolfo; Bouvier, Nicole M; Ito, Kimihito; Medina, Rafael A; Palese, Peter; Krammer, Florian

    2017-04-01

    Infection with influenza virus induces antibodies to the viral surface glycoproteins hemagglutinin and neuraminidase, and these responses can be broadly protective. To assess the breadth and magnitude of antibody responses, we sequentially infected mice, guinea pigs and ferrets with divergent H1N1 or H3N2 subtypes of influenza virus. We measured antibody responses by ELISA of an extensive panel of recombinant glycoproteins representing the viral diversity in nature. Guinea pigs developed high titers of broadly cross-reactive antibodies; mice and ferrets exhibited narrower humoral responses. Then, we compared antibody responses after infection of humans with influenza virus H1N1 or H3N2 and found markedly broad responses and cogent evidence for 'original antigenic sin'. This work will inform the design of universal vaccines against influenza virus and can guide pandemic-preparedness efforts directed against emerging influenza viruses.

  7. Defining the antibody cross-reactome against the influenza virus surface glycoproteins

    Science.gov (United States)

    Nachbagauer, Raffael; Choi, Angela; Hirsh, Ariana; Margine, Irina; Iida, Sayaka; Barrera, Aldo; Ferres, Marcela; Albrecht, Randy A.; García-Sastre, Adolfo; Bouvier, Nicole M.; Ito, Kimihito; Medina, Rafael A.; Palese, Peter; Krammer, Florian

    2017-01-01

    Summary Influenza virus infections induce antibodies against the viral surface glycoproteins hemagglutinin and neuraminidase, and these responses can be broadly protective. To test the breadth and magnitude of antibody responses, mice, guinea pigs and ferrets were sequentially infected with divergent H1N1 or H3N2 viruses. Antibody responses were measured by ELISA against an extensive panel of recombinant glycoproteins representing the viral diversity in nature. Guinea pigs developed high titers of broadly cross-reactive antibodies; mice and ferrets exhibited narrower humoral responses. Then, we compared antibody responses after H1N1 or H3N2 infections in humans and found markedly broad responses and cogent evidence for original antigenic sin. This work will inform universal influenza vaccine design and can guide pandemic preparedness efforts against emerging influenza viruses. PMID:28192418

  8. Milk fat globule membrane glycoproteins: Valuable ingredients for lactic acid bacteria encapsulation?

    Science.gov (United States)

    Guerin, Justine; Burgain, Jennifer; Gomand, Faustine; Scher, Joël; Gaiani, Claire

    2017-10-04

    The membrane (Milk Fat Globule Membrane - MFGM) surrounding the milk fat globule is becoming increasingly studied for its use in food applications due to proven nutritional and technological properties. This review focuses first on current researches which have been led on the MFGM structure and composition and also on laboratory and industrial purification and isolation methods developed in the last few years. The nutritional, health benefits and techno-functional properties of the MFGM are then discussed. Finally, new techno-functional opportunities of MFGM glycoproteins as a possible ingredient for Lactic Acid Bacteria (LAB) encapsulation are detailed. The ability of MFGM to form liposomes entrapping bioactive compounds has been already demonstrated. One drawback is that liposomes are too small to be used for bacteria encapsulation. For the first time, this review points out the numerous advantages to use MFGM glycoproteins as a protecting, encapsulating matrix for bacteria and especially for LAB.

  9. Cyclophosphamide metabolite inducing apoptosis in RLS mouse lymphosarcoma cells is a substrate for P-glycoprotein.

    Science.gov (United States)

    Patutina, O A; Mironova, N L; Logashenko, E B; Popova, N A; Nikolin, V P; Vasil'ev, G V; Kaledin, V I; Zenkova, M A; Vlasov, V V

    2012-01-01

    RLS lymphosarcoma characterized by enhanced expression of mdr1a and mdr1b genes encoding P-glycoprotein is insensitive to low doses of cyclophosphamide, but is susceptible to its high doses approximating the maximum tolerated doses. Induction of apoptotic death of RLS cells by high doses of cyclophosphamide was demonstrated by cytofluorometry and electrophoresis. Experiments on RLS(40) tumor cells derived from RLS lymphosarcoma and characterized by more intensive expression of mdr1a/1b genes showed that the therapeutic effects of cyclophosphamide increased under conditions of simultaneous suppression of these genes by specific small interfering RNA (siRNA). These findings suggest that active cyclophosphamide metabolite can be a substrate for P-glycoprotein.

  10. Similar diagnostic performance for neurocysticercosis of three glycoprotein preparations from Taenia solium metacestodes.

    Science.gov (United States)

    Villota, Guido E; Gomez, Diana I; Volcy, Michel; Franco, Andrés F; Cardona, Edgar A; Isaza, Rodrigo; Sanzón, Fernando; Teale, Judy M; Restrepo, Blanca I

    2003-03-01

    The detection of antibodies to Taenia solium metacestodes is very important in the differential diagnosis of neurocysticercosis (NCC). In this study, an electroimmunotransfer blot (EITB) assay that uses an elaborate protocol with metacestode glycoproteins as antigens was compared with two other Western blots that use glycoproteins obtained using simpler methods, including an eluate from a lectin column, or the vesicular fluid (VF) of the parasite. The concordance between the three assays was 91% in patients with active NCC and 100% in patients with suspected NCC and previous documentation of negative serology. The specificities for the Western blots and the EITB assay were 98% and 100%, respectively (98% concordance). These data suggest that the simplest of these immunoassays, the one that uses the VF of T. solium metacestodes in a Western blot format, can be reliably used for the serologic diagnosis of NCC in developing countries where access to the EITB assay is difficult.

  11. Understanding the Process of Envelope Glycoprotein Incorporation into Virions in Simian and Feline Immunodeficiency Viruses

    Directory of Open Access Journals (Sweden)

    José L. Affranchino

    2014-01-01

    Full Text Available The lentiviral envelope glycoproteins (Env mediate virus entry by interacting with specific receptors present at the cell surface, thereby determining viral tropism and pathogenesis. Therefore, Env incorporation into the virions formed by assembly of the viral Gag polyprotein at the plasma membrane of the infected cells is a key step in the replication cycle of lentiviruses. Besides being useful models of human immunodeficiency virus (HIV infections in humans and valuable tools for developing AIDS therapies and vaccines, simian and feline immunodeficiency viruses (SIV and FIV, respectively are relevant animal retroviruses; the study of which provides important information on how lentiviral replication strategies have evolved. In this review, we discuss the molecular mechanisms underlying the incorporation of the SIV and FIV Env glycoproteins into viral particles.

  12. The pestivirus Erns glycoprotein interacts with E2 in both infected cells and mature virions

    International Nuclear Information System (INIS)

    Lazar, Catalin; Zitzmann, Nicole; Dwek, Raymond A.; Branza-Nichita, Norica

    2003-01-01

    E rns is a pestivirus envelope glycoprotein indispensable for virus attachment and infection of target cells. Unlike the other two envelope proteins E1 and E2, E rns lacks a transmembrane domain and a vast quantity is secreted into the medium of infected cells. The protein is also present in fractions of pure pestivirus virions, raising the important and intriguing question regarding the mechanism of its attachment to the pestivirus envelope. In this study a direct interaction between E rns and E2 glycoproteins was demonstrated in both pestivirus-infected cells and mature virions. By co- and sequential immunoprecipitation we showed that an E rns -E2 heterodimer is assembled very early after translation of the viral polyprotein and before its processing is completed. Our results suggest that E rns is attached to the pestivirus envelope via a direct interaction with E2 and explain the role of E rns in the initial virus-target cell interaction

  13. A novel mechanism of immune evasion mediated by Ebola virus soluble glycoprotein.

    Science.gov (United States)

    Basler, Christopher F

    2013-05-01

    Ebola viruses encode two glycoproteins (GPs): a membrane-associated GP that is present in the viral membrane and mediates viral attachment and entry into host cells; and a secreted, nonstructural glycoprotein (sGP) that is identical to GP over approximately 90% of its length. A recent study by Mohan and colleagues attributes a novel immune evasion mechanism dubbed 'antigenic subversion' to sGP. Using DNA immunization in mice, the authors demonstrate that sGP elicits antibodies that crossreact with GP, but these antibodies are non-neutralizing. Coimmunization with sGP plus GP or sequential immunizations with GP and sGP direct the host antibody response toward non-neutralizing epitopes. Therefore, the production of sGP may prevent effective neutralization of the virus during Ebola virus infection, and may reduce the effectiveness of vaccines that rely upon neutralizing antibody responses.

  14. Characterization of Vesicular Stomatitis Virus Recombinants That Express and Incorporate High Levels of Hepatitis C Virus Glycoproteins

    OpenAIRE

    Buonocore, Linda; Blight, Keril J.; Rice, Charles M.; Rose, John K.

    2002-01-01

    We generated recombinant vesicular stomatitis viruses (VSV) expressing genes encoding hybrid proteins consisting of the extracellular domains of hepatitis C virus (HCV) glycoproteins fused at different positions to the transmembrane and cytoplasmic domains of the VSV G glycoprotein (E1G and E2G). We show that these chimeric proteins are transported to the cell surface and incorporated into VSV virions efficiently. We also generated VSV recombinants in which the gene encoding the VSV G protein...

  15. Human MHC-II with Shared Epitope Motifs Are Optimal Epstein-Barr Virus Glycoprotein 42 Ligands—Relation to Rheumatoid Arthritis

    DEFF Research Database (Denmark)

    Trier, Nicole; Gonzalez-Izarzugaza, Jose Maria; Chailyan, Anna

    2018-01-01

    Rheumatoid arthritis (RA) is a chronic systemic autoimmune disorder of unknown etiology, which is characterized by inflammation in the synovium and joint damage. Although the pathogenesis of RA remains to be determined, a combination of environmental (e.g., viral infections) and genetic factors...... influence disease onset. Especially genetic factors play a vital role in the onset of disease, as the heritability of RA is 50–60%, with the human leukocyte antigen (HLA) alleles accounting for at least 30% of the overall genetic risk. Some HLA-DR alleles encode a conserved sequence of amino acids, referred...... to as the shared epitope (SE) structure. By analyzing the structure of a HLA-DR molecule in complex with Epstein-Barr virus (EBV), the SE motif is suggested to play a vital role in the interaction of MHC II with the viral glycoprotein (gp) 42, an essential entry factor for EBV. EBV has been repeatedly linked to RA...

  16. MicroRNA expression in rainbow trout (Oncorhynchus mykiss) vaccinated with a DNA vaccine encoding the glycoprotein gene of Viral hemorrhagic septicemia virus

    DEFF Research Database (Denmark)

    Bela-Ong, Dennis; Schyth, Brian Dall; Lorenzen, Niels

    particularly to sea-farmed rainbow trout and thus necessitates strategies to mitigate potential disease outbreaks. A DNA vaccine encoding the glycoprotein gene of VHSV has been developed and shown to elicit protective immune responses in laboratory trials. It is important to identify key factors as biomarkers...... during infection and vaccination in order to understand the complex web of interactions involved in the underlying host immune response. Micro ribonucleic acids (miRNAs) are a diverse class of small (18-22 nucleotides) endogenous RNAs that potently mediate post-transcriptional silencing of a wide range...... of genes and are emerging as critical regulators of cellular processes, including immune responses. A microarray experiment in our lab revealed that miR-155, miR-462, and miR-731 were upregulated in fish liver following VHSV infection. Therefore, we analysed the expression of these miRNAs together...

  17. Studying the Impact of Presence of Alpha Acid Glycoprotein and Protein Glycoprotein in Chronic Myeloid Leukemia Patients Treated with Imatinib Mesylate in the State of Qatar

    Directory of Open Access Journals (Sweden)

    Nader I. Ai-Dewik

    2015-01-01

    Full Text Available Despite the efficacy of imatinib mesylate (IM in treating chronic myeloid leukemia (CML, there is a high degree of resistance. Alpha-1-acid glycoprotein may reduce drug efficacy through its ability to interact with IM and blocks it from reaching its target, while protein glycoprotein (PGP may reduce the intracellular concentration of the drug via an active pump mechanism. We thus investigated the correlation between AGP and PGP levels and the resistance/response to treatment. A total of 26 CML patients were investigated for AGP and PGP levels at diagnosis and during treatment. There was no significant difference or correlation between AGP levels and the different groups of patients. There was also no significant difference in the fluorescence intensities of PGP levels among the different patient groups. The resistance observed in our CML patient population could not be correlated with AGP and PGP levels. There was no significant pattern of AGP and PGP expression, irrespective of the response or resistance to treatment.

  18. Complexity explained

    CERN Document Server

    Erdi, Peter

    2008-01-01

    This book explains why complex systems research is important in understanding the structure, function and dynamics of complex natural and social phenomena. Readers will learn the basic concepts and methods of complex system research.

  19. Identification of barley proteins and glycoproteins by various separation techniques and MALDI MS

    Czech Academy of Sciences Publication Activity Database

    Laštovičková, Markéta; Bezouška, Karel; Marchetti, M.; Allmaier, G.; Chmelík, Josef

    2005-01-01

    Roč. 99, S (2005), s307-s309 ISSN 0009-2770. [Meeting on Chemistry and Life /3./. Brno, 20.09.2005-22.09.2005] R&D Projects: GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : glycoprotein * SDS - PAGE * lectin chromatography Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 0.445, year: 2005

  20. The "lecithotrophic" sea urchin Heliocidaris erythrogramma lacks typical yolk platelets and yolk glycoproteins.

    Science.gov (United States)

    Scott, L B; Lennarz, W J; Raff, R A; Wray, G A

    1990-03-01

    The sea urchin Heliocidaris tuberculata undergoes typical development, forming an echinoid pluteus larva, whereas H. erythrogramma undergoes direct development via a highly modified, nonfeeding larva. Using a polyclonal antibody prepared against yolk glycoproteins from the typical developer Stronglyocentrotus purpuratus, we found that H. tuberculata contains cross-reactive proteins in abundance, but H. erythrogramma does not. In addition, we used immunoelectron microscopy to demonstrate that unfertilized eggs of H. tuberculata contain yolk platelets, but those of H. erythrogramma do not.

  1. Refining the Mechanisms of Heniparvirus-Mediated Membrane Fusion Through Mutagenesis of Hendra virus Envelope Glycoproteins

    Science.gov (United States)

    2007-09-06

    glycoprotein single point mutants…..67 Figure 9: Effects of multiple point mutations on fusion activity of HeV F……….……..69 Figure 10: Effects of multiple ...relapsing encephalitis (28). How or whether this latter manifestation of disease is at all analogous to Subacute Sclerosing Panencephalitis (SSPE), a...total DNA per T-25cm2 flask overnight followed by infection with wild-type vaccinia virus (strain WR) at a multiplicity of infection (MOI) of 10. At

  2. Protective efficacy of a recombinant Newcastle disease virus expressing glycoprotein of vesicular stomatitis virus in mice

    OpenAIRE

    Zhang, Minmin; Ge, Jinying; Li, Xiaofang; Chen, Weiye; Wang, Xijun; Wen, Zhiyuan; Bu, Zhigao

    2016-01-01

    Background Vesicular stomatitis virus (VSV) causes severe losses to the animal husbandry industry. In this study, a recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of VSV (rL-VSV-G) was constructed and its pathogenicity and immune protective efficacy in mouse were evaluated. Results In pathogenicity evaluation test, the analysis of the viral distribution in mouse organs and body weight change showed that rL-VSV-G was safe in mice. In immune protection assay, the reco...

  3. Glycoproteins and protein glycations identified in barley grain and malt by 2D-HPLC

    Czech Academy of Sciences Publication Activity Database

    Žídková, Jitka; Petry-Podgorska, Inga; Laštovičková, Markéta; Bobálová, Janette

    2013-01-01

    Roč. 20, č. 1 (2013), s. 43 ISSN 1211-5894. [Discussion in Structural Molecular Biology /11./. 14.03.2013-16.03.2013, Nové Hrady] R&D Projects: GA ČR(CZ) GPP503/12/P395 Institutional support: RVO:68081715 Keywords : barley grain * glycoproteins * 2D-HPLC * MS/MS Subject RIV: CB - Analytical Chemistry, Separation

  4. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb...

  5. A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells

    Science.gov (United States)

    Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2014-01-01

    Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5′-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the production of GDP-l-fucose, the donor substrate required for this process. Initially, we engineered two different insect cell lines to constitutively express Rmd and isolated subclones with fucosylation-negative phenotypes. However, we found the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that this host cell engineering approach is ineffective in insect systems. Thus, we constructed a baculovirus vector designed to express Rmd immediately after infection and facilitate the insertion of genes encoding any glycoprotein of interest for expression later after infection. We used this vector to produce a daughter encoding rituximab and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a nonfucosylated form of this therapeutic antibody. These results indicate that our Rmd+ baculoviral vector can be used to solve the immunogenic core α1,3-fucosylation problem associated with the baculovirus-insect cell system. In conjunction with existing glycoengineered insect cell lines, this vector extends the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production. This new vector also extends the utility of the baculovirus-insect cell system to include the production of recombinant antibodies with enhanced effector functions, due to its ability to block core α1,6-fucosylation. PMID:24362443

  6. Expression and cellular trafficking of GP82 and GP90 glycoproteins during Trypanosoma cruzi metacyclogenesis

    OpenAIRE

    Bayer-Santos, Ethel; Cunha-e-Silva, Narcisa Leal; Yoshida, Nobuko; Franco da Silveira, Jos?

    2013-01-01

    Background: the transformation of noninfective epimastigotes into infective metacyclic trypomastigotes (metacyclogenesis) is a fundamental step in the life cycle of Trypanosoma cruzi, comprising several morphological and biochemical changes. GP82 and GP90 are glycoproteins expressed at the surface of metacyclic trypomastigote, with opposite roles in mammalian cell invasion. GP82 is an adhesin that promotes cell invasion, while GP90 acts as a negative regulator of parasite internalization. Our...

  7. Local expression and exocytosis of viral glycoproteins in multinucleated muscle cells

    OpenAIRE

    1992-01-01

    We have analyzed the distribution of enveloped viral infections in multinucleated L6 muscle cells. A temperature-sensitive vesicular stomatitis virus (mutant VSV ts045) was utilized at the nonpermissive temperature (39 degrees C). As expected, the glycoprotein (G protein) of this mutant was restricted to the ER when the multinucleated cells were maintained at 39 degrees C. We demonstrate that this G protein remained localized when the infection was performed at low dose. By 4 h after infectio...

  8. Extracellular Matrix Glycoprotein-Derived Synthetic Peptides Differentially Modulate Glioma and Sarcoma Cell Migration.

    Science.gov (United States)

    Brösicke, Nicole; Sallouh, Muhammad; Prior, Lisa-Marie; Job, Albert; Weberskirch, Ralf; Faissner, Andreas

    2015-07-01

    Glycoproteins of the extracellular matrix (ECM) regulate proliferation, migration, and differentiation in numerous cell lineages. ECM functions are initiated by small peptide sequences embedded in large constituents that are recognized by specific cellular receptors. In this study, we have investigated the biological effects of peptides derived from collagen type IV and tenascin-C compared to the well-known RGD peptide originally discovered in fibronectin. The influence of glycoproteins and corresponding peptides on the migration of the glioma cell lines U-251-MG and U-373-MG and the sarcoma line S-117 was studied. When the cell lines were tested in a modified Boyden chamber assay on filters coated with the ECM glycoproteins, glioma cells showed a strong migration response on tenascin-C and the basal lamina constituent collagen IV, in contrast to S-117 cells. In order to identify relevant stimulatory motifs, peptides derived from fibronectin (6NHX-GRGDSF), tenascin-C (TN-C, VSWRAPTA), and collagen type IV (MNYYSNS) were compared, either applied in solution in combination with ECM glycoprotein substrates, in solution in the presence of untreated membranes, or coated on the filters of the Boyden chambers. Using this strategy, we could identify the novel tenascin-C-derived peptide motif VSWRAPTA as a migration stimulus for glioma cells. Furthermore, while kin peptides generally blocked the effects of the respective homologous ECM proteins, unexpected effects were observed in heterologous situations. There, in several cases, addition of soluble peptides strongly boosted the response to the coated ECM proteins. We propose that peptides may synergize or antagonize each other by stimulating different signaling pathways.

  9. Highly efficient retrograde gene transfer into motor neurons by a lentiviral vector pseudotyped with fusion glycoprotein.

    Directory of Open Access Journals (Sweden)

    Miyabi Hirano

    Full Text Available The development of gene therapy techniques to introduce transgenes that promote neuronal survival and protection provides effective therapeutic approaches for neurological and neurodegenerative diseases. Intramuscular injection of adenoviral and adeno-associated viral vectors, as well as lentiviral vectors pseudotyped with rabies virus glycoprotein (RV-G, permits gene delivery into motor neurons in animal models for motor neuron diseases. Recently, we developed a vector with highly efficient retrograde gene transfer (HiRet by pseudotyping a human immunodeficiency virus type 1 (HIV-1-based vector with fusion glycoprotein B type (FuG-B or a variant of FuG-B (FuG-B2, in which the cytoplasmic domain of RV-G was replaced by the corresponding part of vesicular stomatitis virus glycoprotein (VSV-G. We have also developed another vector showing neuron-specific retrograde gene transfer (NeuRet with fusion glycoprotein C type, in which the short C-terminal segment of the extracellular domain and transmembrane/cytoplasmic domains of RV-G was substituted with the corresponding regions of VSV-G. These two vectors afford the high efficiency of retrograde gene transfer into different neuronal populations in the brain. Here we investigated the efficiency of the HiRet (with FuG-B2 and NeuRet vectors for retrograde gene transfer into motor neurons in the spinal cord and hindbrain in mice after intramuscular injection and compared it with the efficiency of the RV-G pseudotype of the HIV-1-based vector. The main highlight of our results is that the HiRet vector shows the most efficient retrograde gene transfer into both spinal cord and hindbrain motor neurons, offering its promising use as a gene therapeutic approach for the treatment of motor neuron diseases.

  10. Protein and Site Specificity of Fucosylation in Liver-Secreted Glycoproteins

    Czech Academy of Sciences Publication Activity Database

    Pompach, Petr; Ashline, David J.; Brnáková, Z.; Benicky, J.; Sanda, M.; Goldman, R.

    2014-01-01

    Roč. 13, č. 12 (2014), s. 5561-5569 ISSN 1535-3893 R&D Projects: GA MŠk LH13051; GA ČR GAP206/12/0503 Grant - others:Charles Univ.(CZ) UNCE_204025/2012 Institutional support: RVO:61388971 Keywords : fucose * glycoproteins * liver * site specificity Subject RIV: CE - Biochemistry Impact factor: 4.245, year: 2014

  11. Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies

    OpenAIRE

    Davidson, Edgar; Bryan, Christopher; Fong, Rachel H.; Barnes, Trevor; Pfaff, Jennifer M.; Mabila, Manu; Rucker, Joseph B.; Doranz, Benjamin J.

    2015-01-01

    Cocktails of monoclonal antibodies (MAbs) that target the surface glycoprotein (GP) of Ebola virus (EBOV) are effective in nonhuman primate models and have been used under emergency compassionate-treatment protocols in human patients. However, the amino acids that form the detailed binding epitopes for the MAbs in the ZMapp, ZMAb, and the related MB-003 cocktails have yet to be identified. Other binding properties that define how each MAb functionally interacts with GP—such as affinity, epito...

  12. Comparison of western blot analysis and immunocytochemical detection of P-glycoprotein in multidrug resistant cells.

    OpenAIRE

    Friedlander, M L; Bell, D R; Leary, J; Davey, R A

    1989-01-01

    A sensitive immunocytochemical technique was developed to detect a 170,000 dalton cell membrane glycoprotein (P-gp) in cell lines resistant to vincristine and vinblastine with varying degrees of resistance. P-gp was shown very clearly using the C219 monoclonal antibody and immunocytochemical detection with either antialkaline phosphate or peroxidase-antiperoxidase with silver gold intensification. There was good correlation between the results obtained with immunocytochemical detection of P-g...

  13. The Cloning, Characterization, and Functional Analysis of Murine Pregnancy Specific Glycoproteins

    Science.gov (United States)

    1999-07-23

    Carcinoembryonic antigen gene family members in submandibular salivary gland : demonstration ofpregnancy-specific glycoproteins by cDNA cloning...levels in epithelial carcinomas such as colon cancer and other adenocarcinomas (18, 46). It has been suggested that PSGs may have a role in mediating...site and are later redistributed throughout the myometrium, endometrial stroma, and metrial gland (140-142). In humans, the connective tissue and

  14. A hybrid monolithic column based on boronate-functionalized graphene oxide nanosheets for online specific enrichment of glycoproteins.

    Science.gov (United States)

    Zhou, Chanyuan; Chen, Xiaoman; Du, Zhuo; Li, Gongke; Xiao, Xiaohua; Cai, Zongwei

    2017-05-19

    A hybrid monolithic column based on aminophenylboronic acid (APBA)-functionalized graphene oxide (GO) has been developed and used for selective enrichment of glycoproteins. The APBA/GO composites were homogeneously incorporated into a polymer monolithic column with the help of oligomer matrix and followed by in situ polymerization. The effect of dispersion of APBA/GO composites in the polymerization mixture on the performance of the monolithic column was explored in detail. The presence of graphene oxide not only enlarged the BET surface area from 6.3m 2 /g to 169.4m 2 /g, but also provided abundant boronic acid moieties for glycoprotein extraction, which improved the enrichment selectivity and efficiency for glycoproteins. The APBA/GO hybrid monolithic column was incorporated into a sequential injection system, which facilitated online extraction of proteins. Combining the superior properties of extraordinary surface area of GO and the affinity interaction of APBA to glycoproteins, the APBA/GO hybrid monolithic column showed higher enrichment factors for glycoproteins than other proteins without cis-diol-containing groups. Also, under comparable or even shorter processing time and without the addition of any organic solvent, it showed higher binding capacity toward glycoproteins compared with the conventional boronate affinity monolithic column. The practical applicability of this system was demonstrated by processing of egg white samples for extraction of ovalbumin and ovotransferrin, and satisfactory results were obtained by assay with SDS-PAGE. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Xing Hua Tang

    Full Text Available We investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs.Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.eEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.

  16. Contribution of tumor endothelial cells to drug resistance: anti-angiogenic tyrosine kinase inhibitors act as p-glycoprotein antagonists.

    Science.gov (United States)

    Bani, MariaRosa; Decio, Alessandra; Giavazzi, Raffaella; Ghilardi, Carmen

    2017-05-01

    Tumor endothelial cells (TEC) differ from the normal counterpart, in both gene expression and functionality. TEC may acquire drug resistance, a characteristic that is maintained in vitro. There is evidence that TEC are more resistant to chemotherapeutic drugs, substrates of ATP-binding cassette (ABC) transporters. TEC express p-glycoprotein (encoded by ABCB1), while no difference in other ABC transporters was revealed compared to normal endothelia. A class of tyrosine kinase inhibitors (TKI), used as angiostatic compounds, interferes with the ATPase activity of p-glycoprotein, thus impairing its functionality. The exposure of ovarian adenocarcinoma TEC to the TKIs sunitinib or sorafenib was found to abrogate resistance (proliferation and motility) to doxorubicin and paclitaxel in vitro, increasing intracellular drug accumulation. A similar effect has been reported by the p-glycoprotein inhibitor verapamil. No beneficial effect was observed in combination with cytotoxic drugs that are not p-glycoprotein substrates. The current paper reviews the mechanisms of TEC chemoresistance and shows the role of p-glycoprotein in mediating such resistance. Inhibition of p-glycoprotein by anti-angiogenic TKI might contribute to the beneficial effect of these small molecules, when combined with chemotherapy, in counteracting acquired drug resistance.

  17. IgA antibodies against β2 glycoprotein I in hemodialysis patients are an independent risk factor for mortality.

    Science.gov (United States)

    Serrano, Antonio; García, Florencio; Serrano, Manuel; Ramírez, Elisa; Alfaro, F Javier; Lora, David; de la Cámara, Agustín Gómez; Paz-Artal, Estela; Praga, Manuel; Morales, Jose M

    2012-06-01

    Cardiovascular complications are the most important cause of death in patients on dialysis with end-stage renal disease. Antibodies reacting with β-glycoprotein I seem to play a pathogenic role in antiphospholipid syndrome and stroke and are involved in the origin of atherosclerosis. Here we evaluated the presence of anticardiolipin and anti-β-glycoprotein I antibodies together with other vascular risk factors and their relationship with mortality and cardiovascular morbidity in a cohort of 124 hemodialysis patients prospectively followed for 2 years. Of these, 41 patients were significantly positive for IgA anti-β-glycoprotein I, and the remaining had normal values. At 24 months, overall and cardiovascular mortality and thrombotic events were all significantly higher in patients with high anti-β-glycoprotein I antibodies. Multivariate analysis using Cox regression modeling found that age, hypoalbuminemia, use of dialysis catheters, and IgA β-glycoprotein I antibodies were independent risk factors for death. Thus, IgA antibodies to β-glycoprotein I are detrimental to the clinical outcome of hemodialysis patients.

  18. The changing fate of a secretory glycoprotein in developing maize endosperm.

    Science.gov (United States)

    Arcalis, Elsa; Stadlmann, Johannes; Marcel, Sylvain; Drakakaki, Georgia; Winter, Verena; Rodriguez, Julian; Fischer, Rainer; Altmann, Friedrich; Stoger, Eva

    2010-06-01

    Zeins are the major storage proteins in maize (Zea mays) endosperm, and their accumulation in zein bodies derived from the endoplasmic reticulum is well characterized. In contrast, relatively little is known about post-Golgi compartments or the trafficking of vacuolar proteins in maize endosperm, specifically the presence of globulins in structures resembling protein storage vacuoles that appear in early to mid-stage seed development. We investigated this pathway by expressing and analyzing a recombinant reporter glycoprotein during endosperm maturation, using a combination of microscopy and sensitive glycopeptide analysis. Specific N-glycan acceptor sites on the protein were followed through the stages of grain development, revealing a shift from predominantly paucimannosidic vacuolar glycoforms to predominantly trimmed glycan structures lacking fucose. This was accompanied by a change in the main subcellular localization of the protein from large protein storage vacuole-like post-Golgi organelles to the endoplasmic reticulum and zein bodies. The endogenous storage proteins corn alpha-globulin and corn legumin-1 showed a similar spatiotemporal profile both in transgenic plants expressing the reporter glycoprotein and in wild-type plants. This indicates that the shift of the intracellular trafficking route, as observed with our reporter glycoprotein, may be a common strategy in maize seed development.

  19. Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Xu, Sha; Zhang, Ge-Yuan; Zhang, Huijie; Kitajima, Toshihiko; Nakanishi, Hideki; Gao, Xiao-Dong

    2016-10-21

    To humanize yeast N-glycosylation pathways, genes involved in yeast specific hyper-mannosylation must be disrupted followed by the introduction of genes catalyzing the synthesis, transport, and addition of human sugars. However, deletion of these genes, for instance, OCH1, which initiates hyper-mannosylation, could cause severe defects in cell growth, morphogenesis and response to environmental challenges. In this study, overexpression of RHO1, which encodes the Rho1p small GTPase, is confirmed to partially recover the growth defect of Saccharomyces cerevisiae Δalg3Δoch1 double mutant strain. In addition, transmission electron micrographs indicated that the cell wall structure of RHO1-expressed cells have an enhanced glucan layer and also a recovered mannoprotein layer, revealing the effect of Rho1p GTPase on cell wall biosynthesis. Similar complementation phenotypes have been confirmed by overexpression of the gene that encodes Fks2 protein, a catalytic subunit of a 1,3-β-glucan synthase. Besides the recovery of cell wall structure, the RHO1-overexpressed Δalg3Δoch1 strain also showed improved abilities in temperature tolerance, osmotic potential and drug sensitivity, which were not observed in the Δalg3Δoch1-FKS2 cells. Moreover, RHO1 overexpression could also increase N-glycan site occupancy and the amount of secreted glycoproteins. Overexpression of RHO1 in 'humanized' glycoprotein producing yeasts could significantly facilitate its future industrial applications for the production of therapeutic glycoproteins.

  20. Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule

    Energy Technology Data Exchange (ETDEWEB)

    Yagi, Hirokazu [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan); Nakamura, Masatoshi [National Institute of Agrobiological Sciences, Genetic Resources Conservation Research Unit, Genetic Resources Center (Japan); Yokoyama, Jun [Taiyo Nippon Sanso Corporation, Tsukuba Laboratories (Japan); Zhang, Ying; Yamaguchi, Takumi [National Institutes of Natural Sciences, Institute for Molecular Science and Okazaki Institute for Integrative Bioscience (Japan); Kondo, Sachiko [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan); Kobayashi, Jun [Yamaguchi University, Department of Biological and Environmental Sciences, Faculty of Agriculture (Japan); Kato, Tatsuya; Park, Enoch Y. [Shizuoka University, Laboratory of Biotechnology, Research Institute of Green Science and Technology (Japan); Nakazawa, Shiori [Nagoya University, Sugashima Marine Biological Laboratory, Graduate School of Science (Japan); Hashii, Noritaka; Kawasaki, Nana [National Institute of Health Sciences, Division of Biological Chemistry and Biologicals (Japan); Kato, Koichi, E-mail: kkato@phar.nagoya-cu.ac.jp [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan)

    2015-06-15

    Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing {sup 15}N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a {sup 15}N-enrichment ratio of approximately 80 %. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.

  1. Protein and Glycoprotein Patterns Related to Morphogenesis in Mammillaria gracillis Pfeiff. Tissue Culture

    Directory of Open Access Journals (Sweden)

    Biljana Balen

    2002-01-01

    Full Text Available As plants with Crassulacean Acid Metabolism (CAM, cacti are highly affected by artificial environmental conditions in tissue culture. Plants of Mammillaria gracillis Pfeiff. (Cactaceae propagated in vitro produced callus spontaneously. This habituated callus regenerated normal and hyperhydric shoots without the addition of growth regulators. In order to compare habituated callus with the tumorous one, cactus cells were transformed with two strains of Agrobacterium tumefaciens: the wild strain B6S3 (tumour line TW and the rooty mutant GV3101 (tumour line TR. Gene expression in cactus plants, habituated callus, regenerated shoots and two tumour lines was analysed at the level of cellular and extracellular protein and glycoprotein profiles. Proteins were separated by SDS-polyacrylamide gel electrophoresis and 2-D PAGE electrophoresis and silver stained. Concavalin A-peroxidase staining detected glycoproteins with D-manose in their glycan component on protein blots. Developmentally specific protein patterns of Mammillaria gracillis tissue lines were detected. The 2-D PAGE electrophoresis revealed some tissue specific protein groups. The cellular glycoprotein of 42 kDa detected by ConA was highly expressed in undifferentiated tissues (habituated callus, TW and TR tumours and in hyperhydric regenerants. Tumours produced extracellular proteins of 33, 23 and 22 kDa. The N glycosylation of cellular and extracellular proteins was related to specific developmental stage of cactus tissue.

  2. Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

    International Nuclear Information System (INIS)

    Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing

    2015-01-01

    Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC

  3. Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection

    Energy Technology Data Exchange (ETDEWEB)

    Mahmoud, Nora F. [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Faculty of Pharmacy, Suez Canal University, Ismailia (Egypt); Jasirwan, Chyntia [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Division of Hepatobiliary, Department of Internal Medicine, Faculty of Medicine, University of Indonesia (Indonesia); Kanemoto, Satoshi; Wakata, Aika; Wang, Bochao; Hata, Yuuki [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Nagamata, Satoshi [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Kobe (Japan); Kawabata, Akiko [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Tang, Huamin [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Department of Immunology, Nanjing Medical University, Nanjing (China); Mori, Yasuko, E-mail: ymori@med.kobe-u.ac.jp [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan)

    2016-03-15

    Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. - Highlights: • Glycoprotein B (gB) is highly conserved among herpesviruses. • HHV-6 gB is also abundantly expressed in virions. • In the present study, we showed the function of HHV-6 gB cytoplasmic tail domain (CTD). • We found that deletion of gB CTD impairs the intracellular transport of gB protein to the trans-Golgi network (TGN), and CTD of gB is critical for HHV-6 propagation.

  4. Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection

    International Nuclear Information System (INIS)

    Mahmoud, Nora F.; Jasirwan, Chyntia; Kanemoto, Satoshi; Wakata, Aika; Wang, Bochao; Hata, Yuuki; Nagamata, Satoshi; Kawabata, Akiko; Tang, Huamin; Mori, Yasuko

    2016-01-01

    Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. - Highlights: • Glycoprotein B (gB) is highly conserved among herpesviruses. • HHV-6 gB is also abundantly expressed in virions. • In the present study, we showed the function of HHV-6 gB cytoplasmic tail domain (CTD). • We found that deletion of gB CTD impairs the intracellular transport of gB protein to the trans-Golgi network (TGN), and CTD of gB is critical for HHV-6 propagation.

  5. Carbohydrates of influenza virus. I. Glycopeptides derived from viral glycoproteins after labeling with radioactive sugars

    International Nuclear Information System (INIS)

    Schwarz, R.T.; Schmidt, M.F.G.; Anwer, U.; Klenk, H.D.

    1977-01-01

    The carbohydrate moiety of the influenza glycoproteins NA, HA 1 , and HA 2 were analyzed by labeling with radioactive sugars. Analysis of glycopeptides obtained after digestion with Pronase indicated that there are at least two different types of carbohydrate side chains. The side chain of type I is composed of glucosamine, mannose, galactose, and fucose. It is found on NA, HA 1 , and HA 2 . The side chain of type II contains a high amount of mannose and is found only on NA and HA 2 . The molecular weights of the corresponding glycopeptides obtained from virus grown in chicken ambryo cells are 2,600 for type I and 2,000 for type II. The glycoproteins of virus grown in MDBK cells have a higher molecular weight than those of virus grown in chicken embryo cells, and there is a corresponding difference in the molecular weights of the glycopeptides. Under conditions of partial inhibition of glycosylation, virus particles were isolated that contained hemagglutinin with reduced carbohydrate content. Glycopeptide analysis indicated that this reduction is due to the lack of whole carbohydrate side chains and not to the incorporation of incomplete ones. This observation suggests that glycosylation of the viral glycoproteins involves en bloc transfer of the core sugars to the polypeptide chains

  6. Glycosylation of dengue virus glycoproteins and their interactions with carbohydrate receptors: possible targets for antiviral therapy.

    Science.gov (United States)

    Idris, Fakhriedzwan; Muharram, Siti Hanna; Diah, Suwarni

    2016-07-01

    Dengue virus, an RNA virus belonging to the genus Flavivirus, affects 50 million individuals annually, and approximately 500,000-1,000,000 of these infections lead to dengue hemorrhagic fever or dengue shock syndrome. With no licensed vaccine or specific antiviral treatments available to prevent dengue infection, dengue is considered a major public health problem in subtropical and tropical regions. The virus, like other enveloped viruses, uses the host's cellular enzymes to synthesize its structural (C, E, and prM/M) and nonstructural proteins (NS1-5) and, subsequently, to glycosylate these proteins to produce complete and functional glycoproteins. The structural glycoproteins, specifically the E protein, are known to interact with the host's carbohydrate receptors through the viral proteins' N-glycosylation sites and thus mediate the viral invasion of cells. This review focuses on the involvement of dengue glycoproteins in the course of infection and the virus' exploitation of the host's glycans, especially the interactions between host receptors and carbohydrate moieties. We also discuss the recent developments in antiviral therapies that target these processes and interactions, focusing specifically on the use of carbohydrate-binding agents derived from plants, commonly known as lectins, to inhibit the progression of infection.

  7. A lectin-based gold nanoparticle assay for probing glycosylation of glycoproteins.

    Science.gov (United States)

    Sánchez-Pomales, Germarie; Morris, Todd A; Falabella, James B; Tarlov, Michael J; Zangmeister, Rebecca A

    2012-09-01

    We report a glycoanalysis method in which lectins are used to probe the glycans of therapeutic glycoproteins that are adsorbed on gold nanoparticles. A model mannose-presenting glycoprotein, ribonuclease B (RNase B), and the therapeutic monoclonal antibody (mAb) rituximab, were found to adsorb spontaneously and non-specifically to bare gold nanoparticles such that glycans were accessible for lectin binding. Addition of a multivalent binding lectin, such as concanavalin A (Con A), to a solution of the modified gold nanoparticles resulted in cross-linking of the nanoparticles. This phenomenon was evidenced within 1 min by a change in the hydrodynamic diameter, D(H), measured by dynamic light scattering (DLS) and a shift and increase in absorbance of the plasmon resonance band of the gold nanoparticles. By combining the sugar-binding specificity and the cross-linking capabilities of lectins, the non-specific adsorption of glycoproteins to gold surfaces, and the unique optical reporting properties of gold nanoparticles, a glycosylation pattern of rituximab could be generated. This assay provides advantages over currently used glycoanalysis methods in terms of short analysis time, simplicity of the conjugation method, convenience of simple spectroscopic detection, and feasibility of providing glycan characterization of the protein drug product by using a variety of binding lectins. Copyright © 2012 Wiley Periodicals, Inc.

  8. Complex chemistry

    International Nuclear Information System (INIS)

    Kim, Bong Gon; Kim, Jae Sang; Kim, Jin Eun; Lee, Boo Yeon

    2006-06-01

    This book introduces complex chemistry with ten chapters, which include development of complex chemistry on history coordination theory and Warner's coordination theory and new development of complex chemistry, nomenclature on complex with conception and define, chemical formula on coordination compound, symbol of stereochemistry, stereo structure and isomerism, electron structure and bond theory on complex, structure of complex like NMR and XAFS, balance and reaction on solution, an organo-metallic chemistry, biology inorganic chemistry, material chemistry of complex, design of complex and calculation chemistry.

  9. Carbohydrate phenotyping of human and animal milk glycoproteins.

    Science.gov (United States)

    Gustafsson, Anki; Kacskovics, Imre; Breimer, Michael E; Hammarström, Lennart; Holgersson, Jan

    2005-03-01

    Breast-milk has a well-known anti-microbial effect, which is in part due to the many different carbohydrate structures expressed. This renders it a position as a potential therapeutic for treatment of infection by different pathogens, thus avoiding the drawbacks of many antibiotics. The plethora of carbohydrate epitopes in breast-milk is known to differ between species, with human milk expressing the most complex one. We have investigated the expression of protein-bound carbohydrate epitopes in milk from man, cow, goat, sheep, pig, horse, dromedary and rabbit. Proteins were separated by SDS-PAGE and the presence of carbohydrate epitopes on milk proteins were analysed by Western blotting using different lectins and carbohydrate-specific antibodies. We show that ABH, Lewis (Le)x, sialyl-Lex, Lea, sialyl-Lea and Leb carbohydrate epitopes are expressed mainly on man, pig and horse milk proteins. The blood group precursor structure H type 1 is expressed in all species investigated, while only pig, dromedary and rabbit milk proteins carry H type 2 epitopes. These epitopes are receptors for Helicobacter pylori (Leb and sialyl-Lex), enteropathogenic (H type 1, Lea and Lex) and enterotoxic Escherichia coli (heat-stable toxin; H type 1 and 2), and Campylobacter jejuni (H type 2). Thus, milk from these animals or their genetically modified descendants could have a therapeutic effect by inhibiting pathogen colonization and infection.

  10. Myelin oligodendrocyte glycoprotein and aquaporin-4 antibodies are highly specific in children with acquired demyelinating syndromes.

    Science.gov (United States)

    Duignan, Sophie; Wright, Sukhvir; Rossor, Tom; Cazabon, John; Gilmour, Kimberly; Ciccarelli, Olga; Wassmer, Evangeline; Lim, Ming; Hemingway, Cheryl; Hacohen, Yael

    2018-02-22

    Our objectives were to evaluate the utility of measuring myelin oligodendrocyte glycoprotein (MOG) and aquaporin-4 (AQP4) antibodies (Ab) in clinical practice and describe their associated neurological phenotypes in children. Between 2012 and 2017, 371 children with suspected acquired demyelinating syndromes (ADS) seen in three tertiary centres were tested for MOG-Ab and AQP4-Ab. Medical notes were retrospectively reviewed, and clinical and demographic data compiled. Clinical phenotyping was performed blinded to the antibody results. After review, 237 of the 371 were diagnosed with ADS. Of these, 76 out of 237 (32.1%) were MOG-Ab positive and 14 out of 237 (5.9%) were AQP4-Ab positive. None were positive for both autoantibodies. All 134 patients with non-ADS were negative for MOG-Ab. MOG-Ab were identified in 45 out of 70 (64.3%) patients presenting with acute disseminated encephalomyelitis (ADEM) and in 24 out of 25 patients with relapsing ADEM. Thirty-six out of 75 (48%) MOG-Ab positive patients relapsed. Of the 33 children with neuromyelitis optic spectrum disorder, 14 were AQP4-Ab positive, 13 were MOG-Ab positive, and 6 were seronegative. Of the children with longitudinal samples, 8 out of 13 AQP4-Ab remained positive during the disease course compared to 35 out of 43 MOG-Ab (13/16 monophasic and 22/27 relapsing). Myelin oligodendrocyte glycoprotein antibodies were identified in a third of children with ADS. Almost half of the MOG-Ab positive children relapsed and the majority of them remained antibody positive over 4-years follow-up. Myelin oligodendrocyte glycoprotein antibodies (MOG-Ab) are highly specific for acquired demyelinating syndromes (ADS). Myelin oligodendrocyte glycoprotein antibodies are not identified in children with peripheral demyelination or genetic leukodystrophies/hypomyelination. Up to 48% of MOG-Ab ADS paediatric patients relapse, higher than previously thought. Seroconversion to MOG-Ab negative status is infrequent; patients may test

  11. Epstein–Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus

    Energy Technology Data Exchange (ETDEWEB)

    Changotra, Harish; Turk, Susan M. [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA (United States); Artigues, Antonio [Department of Biochemistry, University of Kansas Medical Center, Kansas City, KS (United States); Thakur, Nagendra; Gore, Mindy; Muggeridge, Martin I. [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA (United States); Hutt-Fletcher, Lindsey M., E-mail: lhuttf@lsuhsc.edu [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA (United States)

    2016-02-15

    The Epstein–Barr virus glycoprotein complex gMgN has been implicated in assembly and release of fully enveloped virus, although the precise role that it plays has not been elucidated. We report here that the long predicted cytoplasmic tail of gM is not required for complex formation and that it interacts with the cellular protein p32, which has been reported to be involved in nuclear egress of human cytomegalovirus and herpes simplex virus. Although redistribution of p32 and colocalization with gM was not observed in virus infected cells, knockdown of p32 expression by siRNA or lentivirus-delivered shRNA recapitulated the phenotype of a virus lacking expression of gNgM. A proportion of virus released from cells sedimented with characteristics of virus lacking an intact envelope and there was an increase in virus trapped in nuclear condensed chromatin. The observations suggest the possibility that p32 may also be involved in nuclear egress of Epstein–Barr virus. - Highlights: • The predicted cytoplasmic tail of gM is not required to complex with gN. • Cellular p32 can interact with the predicted cytoplasmic tail of EBV gM. • Knockdown of p32 recapitulates the phenotype of virus lacking the gNgM complex.

  12. Epstein–Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus

    International Nuclear Information System (INIS)

    Changotra, Harish; Turk, Susan M.; Artigues, Antonio; Thakur, Nagendra; Gore, Mindy; Muggeridge, Martin I.; Hutt-Fletcher, Lindsey M.

    2016-01-01

    The Epstein–Barr virus glycoprotein complex gMgN has been implicated in assembly and release of fully enveloped virus, although the precise role that it plays has not been elucidated. We report here that the long predicted cytoplasmic tail of gM is not required for complex formation and that it interacts with the cellular protein p32, which has been reported to be involved in nuclear egress of human cytomegalovirus and herpes simplex virus. Although redistribution of p32 and colocalization with gM was not observed in virus infected cells, knockdown of p32 expression by siRNA or lentivirus-delivered shRNA recapitulated the phenotype of a virus lacking expression of gNgM. A proportion of virus released from cells sedimented with characteristics of virus lacking an intact envelope and there was an increase in virus trapped in nuclear condensed chromatin. The observations suggest the possibility that p32 may also be involved in nuclear egress of Epstein–Barr virus. - Highlights: • The predicted cytoplasmic tail of gM is not required to complex with gN. • Cellular p32 can interact with the predicted cytoplasmic tail of EBV gM. • Knockdown of p32 recapitulates the phenotype of virus lacking the gNgM complex.

  13. Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies

    NARCIS (Netherlands)

    Ikoma, M; Liljeqvist, JA; Glazenburg, KL; The, TH; Welling-Wester, S; Groen, J.

    Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of

  14. Decreased levels of alpha-1-acid glycoprotein are related to the mortality of septic patients in the emergency department

    Directory of Open Access Journals (Sweden)

    Romualdo Barroso-Sousa

    2013-01-01

    Full Text Available OBJECTIVE: To determine the validity of alpha-1-acid glycoprotein as a novel biomarker for mortality in patients with severe sepsis. METHODS: We prospectively included patients with severe sepsis or septic shock at the emergency department at a single tertiary referral teaching hospital. All of the patients were enrolled within the first 24 hours of emergency department admission, and clinical data and blood samples were obtained. As the primary outcome, we investigated the association of serum levels of alpha-1-acid glycoprotein and 96-hour mortality with logistic regression analysis and generalized estimating equations adjusted for age, sex, shock status and Acute Physiology and Chronic Health Evaluation II score. RESULTS: Patients with septic shock had lower alpha-1-acid glycoprotein levels at the time of emergency department admission compared to patients without shock (respectively, 149.1 ±42.7 vs. 189.8 ±68.6; p = 0.005. Similarly, non-survivors in the first 96 hours were also characterized by lower levels of alpha-1-acid glycoprotein at the time of emergency department admission compared to survivors (respectively, 132.18 ±50.2 vs. 179.8 ±61.4; p = 0.01. In an adjusted analysis, alpha-1-acid glycoprotein levels ≤120 mg/dL were significantly associated with 96-hour mortality (odds ratio = 14.37; 95% confidence interval = 1.58 to 130.21. CONCLUSION: Septic shock patients exhibited lower circulating alpha-1-acid glycoprotein levels than patients without shock. Alpha-1-acid glycoprotein levels were independently associated with 96-hour mortality in individuals with severe sepsis.

  15. Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins

    Directory of Open Access Journals (Sweden)

    Yadvinder S. Ahi

    2016-11-01

    Full Text Available Gammaretroviruses, such as murine leukemia viruses (MLVs, encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called “glycogag” (glycosylated Gag. MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes.

  16. Comparison of the prognosis of spontaneous and percutaneous coronary intervention-related myocardial infarction.

    Science.gov (United States)

    Leonardi, Sergio; Thomas, Laine; Neely, Megan L; Tricoci, Pierluigi; Lopes, Renato D; White, Harvey D; Armstrong, Paul W; Giugliano, Robert P; Antman, Elliott M; Califf, Robert M; Newby, L Kristin; Mahaffey, Kenneth W

    2012-12-04

    This study compared prognoses of myocardial infarction related to percutaneous coronary intervention (PCI, procedural MI) using increasing creatine kinase-myocardial band (CK-MB) thresholds with spontaneous MI. Procedural MI usually is defined by a CK-MB elevation of more than 3 times the upper limit of normal (ULN), but higher thresholds have been proposed. Patients from the EARLY-ACS (Early Glycoprotein IIb/IIIa Inhibition in Non-ST-Segment Elevation Acute Coronary Syndrome) study and the SYNERGY (Superior Yield of the New strategy of Enoxaparin, Revascularization and GlYcoprotein IIb/IIIa inhibitors) study treated with PCI were included. The primary end point was 1-year all-cause mortality from 24 h after PCI. To determine an enzymatic threshold for procedural MI with a prognosis similar to that of spontaneous MI, we redefined procedural MI using increasing CK-MB thresholds and compared corresponding hazard ratios with those of spontaneous MI (CK-MB more than twice the ULN). Hazard ratios for mortality for procedural and spontaneous MI were calculated using Cox proportional hazards regression and Global Registry of Acute Cardiac Events covariates for risk adjustment. Nine thousand eighty-seven patients who underwent PCI (46.8%) were included; 773 procedural MI and 239 spontaneous MI occurred within 30 days. Adjusted hazard ratios for 1-year death were 1.39 (95% confidence interval [CI]: 1.01 to 1.89) for procedural MI and 5.37 (95% CI: 3.90 to 7.38) for spontaneous MI. The CK-MB threshold for procedural MI that achieved the same prognosis as spontaneous MI was 27.7 times the ULN (95% CI: 13.9 to 58.4), but this differed between the SYNERGY study (57.9 times the ULN, 95% CI: 17.9 to 63.6) and the EARLY-ACS study (20.4 times the ULN, 95% CI: 5.16 to 24.2). Of all procedural MI, 49 (6%) had CK-MB elevations of 27.7 or more times the ULN. The current enzymatic definition of procedural MI (CK-MB more than 3 times the ULN) used in clinical trials is less strongly

  17. An analysis of the Association of Society of Chest Pain Centers Accreditation to American College of Cardiology/American Heart Association non-ST-segment elevation myocardial infarction guideline adherence.

    Science.gov (United States)

    Chandra, Abhinav; Glickman, Seth W; Ou, Fang-Shu; Peacock, W Frank; McCord, James K; Cairns, Charles B; Peterson, Eric D; Ohman, E Magnus; Gibler, W Brian; Roe, Matthew T

    2009-07-01

    Since 2003, the Society of Chest Pain Centers (SCPC) has provided hospital accreditation for acute coronary syndrome care processes. Our objective is to evaluate the association between SCPC accreditation and adherence to the American College of Cardiology/American Heart Association (ACC/AHA) evidence-based guidelines for non-ST-segment elevation myocardial infarction (NSTEMI). The secondary objective is to describe the clinical outcomes and the association with accreditation. We conducted a secondary analysis of data from patients with NSTEMI enrolled in the Can Rapid Risk Stratification of Unstable Angina Patients Suppress Adverse Outcomes With Early Implementation of the ACC/AHA Guidelines (CRUSADE) quality improvement initiative in 2005. The analysis explored differences between SCPC-accredited and nonaccredited hospitals in evidence-based therapy given within the first 24 hours (including aspirin, beta-blocker, glycoprotein IIb/IIIa inhibitors, heparin, and ECG within 10 minutes). Of 33,238 patients treated at 21 accredited hospitals and 323 nonaccredited hospitals, those at SCPC-accredited centers (n=3,059) were more likely to receive aspirin (98.1% versus 95.8%; odds ratio [OR] 1.73; 95% confidence interval [CI] 1.06 to 2.83) and beta-blockers (93.4% versus 90.6%; OR 1.68; 95% CI 1.04 to 2.70) within 24 hours than patients at non-SCPC-accredited centers (n=30,179). No difference was observed in obtaining a timely ECG (40.4% versus 35.2%; OR 1.28; 95% CI 0.98 to 1.67), administering a glycoprotein IIb/IIIa inhibitor (OR 1.30; 95% CI 0.93 to 1.80), or administering heparin (OR 1.12; 95% CI 0.74 to 1.70). Also, there was no significant difference in risk-adjusted mortality for patients treated at SCPC hospitals versus nonaccredited hospitals (3.4% versus 3.5%; adjusted OR 1.17; 95% CI 0.88 to 1.55). SCPC-accredited hospitals had higher NSTEMI ACC/AHA evidence-based guideline adherence in the first 24 hours of care on 2 of the 5 measures. No difference in

  18. [Incorporation of glycoproteins of the Aujeszky's disease virus ( Suid herpesvirus 1) into artificial liposome membranes and their interaction with cells].

    Science.gov (United States)

    Vrublevskaia, V V; Vinokurov, M G; Kholodkov, O A; Kornev, A N; Morenkov, O S

    2004-01-01

    The purpose of the case study was to investigate the interplay between liposomes, containing the in-built glycoproteins of the Aujeszky disease virus (ADV, Suid herpesvirus 1) with plasmatic membranes of sensitive cells. The conditions of reconstructing the ADV glycoproteins into artificial-liposome membranes were optimized. The above liposomes (virosomes), 40 x 200 nm, were impermeable to univalent ions, which confirmed the virosome membranes were intact. The gE and gB glycoproteins (90-98% of them) were located, inside the liposome membrane with the outwards orientation of their ecto-domain fragments. Virosomes were binding with cells in the dose-dependent mode. The purified ADV virions, the ADV gB glycoproteins and heparin inhibited such binding process of virosomes with cells, which denoted the specificity of their interaction with cells. An effective internalization of cell-binding virosomes was observed at 37 degrees C. The conclusion is that the ADV glycoproteins, constructed into the liposome membranes, simulate adequately enough the viral receptor structures and that the thus obtained virosomes could be used to investigate the interplay between alpha-herpes viruses and cells.

  19. Protective effect of glycoprotein isolated from Ulmus davidiana Nakai on carbon tetrachloride-induced mouse liver injury.

    Science.gov (United States)

    Lee, Sei-Jung; Oh, Phil-Sun; Ko, Jeong-Hyeon; Lim, Kwang; Lim, Kye-Taek

    2006-01-01

    This study was carried out to evaluate the hepatoprotective activity of glycoprotein isolated from the stems of Ulmus davidiana Nakai (UDN), which has been used as an anti-inflammatory agent in folk medicine. We evaluated lipid peroxidation in glucose/glucose oxidase (G/GO)-induced BNL CL.2 cells and measured thiobarbituric acid reactive substances (TBARS), lactate dehydrogenase (LDH), nitric oxide (NO), antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)), activity of cytotoxic-related signals (hepatic cytochrome c, nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1)) and levels of plasma lipids (triglyceride (TG) and total cholesterol (TC)) in carbon tetrachloride (CCl(4,) 1.0 mL kg(-1))-induced A/J mouse. The results in G/GO-induced BNL CL.2 cells showed that UDN glycoprotein had a dose-dependent inhibitory effect on lipid peroxidation. The results in carbon tetrachloride (CCl(4,) 1.0 mL kg(-1))-induced A/J mouse indicated that treatment with UDN glycoprotein (40 mg kg -1) lowered LDH activity and TBARS formation, and increased NO production and antioxidant enzymes activity, compared with control. Also, our finding from CCl(4)-treated mice after pretreatment with UDN glycoprotein demonstrated that the activity of cytotoxic-related signals decreased but the levels of plasma lipids increased, compared with CCl(4) treatment alone. Here, we speculate that UDN glycoprotein has a protective character to CCl(4)-induced mouse liver injury.

  20. Profiling of Concanavalin A-Binding Glycoproteins in Human Hepatic Stellate Cells Activated with Transforming Growth Factor-β1

    Directory of Open Access Journals (Sweden)

    Yannan Qin

    2014-11-01

    Full Text Available Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA was increased in human hepatic stellate cells (HSCs following activation by transforming growth factor-β1 (TGF-β1; however, little is known about the ConA-binding glycoproteins (CBGs of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-β1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-2 [PLCB2] were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

  1. A Study of Anti Beta-2 Glycoprotein I and Anti-Prothrombin Antibodies in Patients with Unexplained Recurrent Pregnancy Losses.

    Science.gov (United States)

    Singh, Angad; Nangia, Anita; Sharma, Sunita; Puri, Manju

    2016-06-01

    To compare the levels of IgG and IgM anti beta-2 glycoprotein I antibodies and IgG and IgM anti prothrombin antibodies among women with unexplained recurrent pregnancy losses and women with at least 2 live issues. To compare the prevalence of newer anti beta-2 glycoprotein I & anti prothrombin antibodies with conventional Lupus anticoagulant & anticardiolipin antibodies. 50 women with recurrent pregnancy losses & 50 matched controls were evaluated for the presence of: Lupus anticoagulant-screened by LA sensitive aPTT& DRVV and confirmatory Staclot Assay. ELISA kits were used for detecting IgG & IgM anticardiolipin, anti beta-2 glycoprotein I & anti prothrombin antibodies. 11/50 (22 %) women in study group and none in control group had circulating antiphospholipid antibodies. 2 cases (4 %) had lupus anticoagulant. 1 case (2 %) had anticardiolipin antibody & 6 cases (12 %) were positive for anti beta-2 Glycoprotein I antibody (p value = 0.027). 3 cases (6 %) had anti prothrombin antibody. All were mutually exclusive except for one. Women with recurrent pregnancy losses should be tested for anti beta-2 Glycoprotein I antibodies & anti prothrombin antibodies in addition to conventional lupus anticoagulant and anticardiolipin antibodies. This approach can decrease the incidence of SNAP (seronegative antiphospholipid syndrome) cases while establishing the true prevalence of antiphospholipid syndrome.

  2. Induction of apoptosis and reversal of permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM by ginsenoside Rh2

    Science.gov (United States)

    Zhang, Hui; Gong, Jian; Zhang, Huilai; Kong, Di

    2015-01-01

    Multidrug resistance is a phenomenon that cancer cells develop a cross-resistant phenotype against several unrelated drugs, and permeability glycoprotein derived from the overexpression of multidrug resistance gene 1 has been taken as the most significant cause of multidrug resistance. In the present study, ginsenoside Rh2 was used to reverse permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM cell line. Effects of ginsenoside Rh2 on the apoptotic process and caspase-3 activity of MCF-7 and MCF-7/ADM cell lines were determined using flow cytometry and microplate reader. Methyl thiazolyl tetrazolium test was conducted to assess the IC50 values of ginsenoside Rh2 and adriamycin on MCF-7 and MCF-7/ADM cultures; Rhodamin 123 assay was used to assess the retention of permeability glycoprotein after ginsenoside Rh2 treatment; flow cytometry and real time polymerase chain reaction were used to determine the expression levels of permeability glycoprotein and multidrug resistance gene 1 in drug-resistant cells and their parental cells after exposure to ginsenoside Rh2. The results showed that ginsenoside Rh2, except for inducing apoptosis, had the ability to reverse multidrug resistance in MCF-7/ADM cell line without changing the expression levels of permeability glycoprotein and multidrug resistance gene 1. Our findings provided some valuable information for the application of ginsenoside Rh2 in cancer therapy, especially for multidrug resistance reversal in clinic. PMID:26191135

  3. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    International Nuclear Information System (INIS)

    Grose, C.; Jackson, W.; Traugh, J.A.

    1989-01-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [γ- 32 P]ATP. The same glycoprotein was phosphorylated when [ 32 P]GTP was substituted for [ 32 P]ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein

  4. Recombinant pestivirus E2 glycoproteins prevent viral attachment to permissive and non permissive cells with different efficiency.

    Science.gov (United States)

    Asfor, A S; Wakeley, P R; Drew, T W; Paton, D J

    2014-08-30

    Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen, which like other pestiviruses has similar molecular biological features to hepaciviruses, including human Hepatitis C virus. The pestivirus E2 glycoproteins are the major target for virus-neutralising antibodies, as well as playing a role in receptor binding and host range restriction. In this study, recombinant E2 glycoproteins (rE2) derived from three different pestivirus species were examined for their inhibitory effects on pestivirus infectivity in cell culture. Histidine-tagged rE2 glycoproteins of BVDV type 2 strain 178003, BVDV type 1 strain Oregon C24V and CSFV strain Alfort 187 were produced in Spodoptera frugiperda insect cells and purified under native conditions. The ability of rE2 glycoprotein to inhibit the infection of permissive cells by both homologous and heterologous virus was compared, revealing that the inhibitory effects of rE2 glycoproteins correlated with the predicted similarity of the E2 structures in the recombinant protein and the test virus. This result suggests that the sequence and structure of E2 are likely to be involved in the host specificity of pestiviruses at their point of uptake into cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Complex Narratives

    NARCIS (Netherlands)

    Simons, J.; Buckland, W.

    2014-01-01

    In the opening chapter, "Complex Narratives," Jan Simons brings together narratology, game theory, and complexity theory to untangle the intricate nature of complex narratives in contemporary cinema. He presents an overview of the different concepts - forking path narratives, mind-game films,

  6. phenanthroline complex

    Indian Academy of Sciences (India)

    ABHRANIL DE

    2018-02-28

    Feb 28, 2018 ... complex in a unique binding motif and provide additional stability to the compound in the solid state. This iron(II) complex is able to catalyze the cleavage of aromatic C-C linkage of 2,5-dihydroxybenzoic acid (Gentisic acid,. GA) in oxygen environment. The iron(II) complex in the presence of two equivalent ...

  7. (II) complexes

    African Journals Online (AJOL)

    activities of Schiff base tin (II) complexes. Neelofar1 ... Conclusion: All synthesized Schiff bases and their Tin (II) complexes showed high antimicrobial and ...... Singh HL. Synthesis and characterization of tin (II) complexes of fluorinated Schiff bases derived from amino acids. Spectrochim Acta Part A: Molec Biomolec.

  8. Interaction of the recently approved anticancer drug nintedanib with human acute phase reactant α 1-acid glycoprotein

    Science.gov (United States)

    Abdelhameed, Ali Saber; Ajmal, Mohammad Rehan; Ponnusamy, Kalaiarasan; Subbarao, Naidu; Khan, Rizwan Hasan

    2016-07-01

    A comprehensive study of the interaction of the newly approved tyrosine kinase inhibitor, Nintedanib (NTB) and Alpha-1 Acid Glycoprotein (AAG) has been carried out by utilizing UV-Vis spectroscopy, fluorescence spectroscopy, circular dichroism, dynamic light scattering and molecular docking techniques. The obtained results showed enhancement of the UV-Vis peak of the protein upon binding to NTB with the fluorescence intensity of AAG is being quenched by NTB via the formation of ground state complex (i.e. Static quenching). Forster distance (Ro) obtained from fluorescence resonance energy transfer (FRET) is found to be 2.3 nm. The calculated binding parameters from the modified Stern-Volmer equation showed that NTB binds to AAG with a binding constant in the order of 103. Conformational alteration of the protein upon its binding to NTB was confirmed by the circular dichroism. Dynamic light scattering results showed that the binding interaction of NTB leads to the reduction in hydrodynamic radii of AAG. Dynamic molecular docking results showed that the NTB fits into the central binding cavity in AAG and hydrophobic interaction played the key role in the binding process also the docking studies were performed with methotrexate and clofarabine drugs to look into the common binding regions of these drugs on AAG molecule, it was found that five amino acid residues namely Phe 113, Arg 89, Tyr 126, Phe 48 and Glu 63 were common among the binding regions of three studied drugs this phenomenon of overlapping binding regions may influence the drug transport by the carrier molecule in turn affecting the metabolism of the drug and treatment outcome.

  9. Physicochemical Characteristics and Anti-Inflammatory Activities of Antrodan, a Novel Glycoprotein Isolated from Antrodia cinnamomea Mycelia

    Directory of Open Access Journals (Sweden)

    Chun-Hung Chiu

    2013-12-01

    Full Text Available Antrodia cinnamomea (AC is a unique fungus found inhabiting the rotten wood of Cinnamomum kanehirai. A submerged liquid culture of AC has been developed and its bioproducts have been used to meet the market demand for natural fruiting bodies. AC exhibits anti-inflammatory, antitumor, antioxidant, and immunomodulatory effects. Previously, we isolated polysaccharide AC-2 from AC mycelia by means of alkali extraction with subsequent acid precipitation and found it had a pronounced anti-inflammatory effect. In this study, a novel polysaccharide named “antrodan” was obtained by further purification of AC-2 using Sepharose CL-6B column chromatography. Antrodan exhibited a molecular weight of 442 kD and contained a particularly high content of uronic acid (152.6 ± 0.8 mg/g. The protein content was 71.0%, apparently, higher than the carbohydrate content (14.1%, and thus antrodan was characterized as a glycoprotein. Its total glucan content was 15.65%, in which β-glucan (14.20% was prominently higher than α-glucan (1.45%. Its FTIR confirmed the presence of β-linkages between sugars, and intramolecular amide bonds between sugars and amino acids. Its 1H-NMR spectrum showed that antrodan was a complex union of α- and β-glucans, which had (1→ 4-linked α-Glcp and (1→ 3-linked β-Glcp linkages to the carbohydrate chains via asparagine linked to protein site. Biologically, antrodan was confirmed to be totally non-detrimental to RAW 264.7 cell line even at dose as high as 400 μg/mL. It showed potent suppressing effect on the lipopolysaccharide-induced inflammatory responses in RAW 264.7 cell line. Moreover, antrodan significantly reduced the nitrogen oxide production at doses as low as 18.75 μg/mL.

  10. Interaction of anticancer drug clofarabine with human serum albumin and human α-1 acid glycoprotein. Spectroscopic and molecular docking approach.

    Science.gov (United States)

    Ajmal, Mohammad Rehan; Nusrat, Saima; Alam, Parvez; Zaidi, Nida; Khan, Mohsin Vahid; Zaman, Masihuz; Shahein, Yasser E; Mahmoud, Mohamed H; Badr, Gamal; Khan, Rizwan Hasan

    2017-02-20

    The binding interaction between clofarabine, an important anticancer drug and two important carrier proteins found abundantly in human plasma, Human Serum Albumin (HSA) and α-1 acid glycoprotein (AAG) was investigated by spectroscopic and molecular modeling methods. The results obtained from fluorescence quenching experiments demonstrated that the fluorescence intensity of HSA and AAG is quenched by clofarabine and the static mode of fluorescence quenching is operative. UV-vis spectroscopy deciphered the formation of ground state complex between anticancer drug and the two studied proteins. Clofarabine was found to bind at 298K with both AAG and HSA with the binding constant of 8.128×10 3 and 4.120×10 3 for AAG and HSA, respectively. There is stronger interaction of clofarabine with AAG as compared to HSA. The Gibbs free energy change was found to be negative for the interaction of clofarabine with AAG and HSA indicating that the binding process is spontaneous. Binding of clofarabine with HSA and AAG induced ordered structures in both proteins and lead to molecular compaction. Clofarabine binds to HSA near to drug site II. Hydrogen bonding and hydrophobic interactions were the main bonding forces between HSA-clofarabine and AAG-clofarabine as revealed by docking results. This study suggests the importance of binding of anticancer drug to AAG spatially in the diseases like cancers where the plasma concentration of AAG increases many folds. Design of drug dosage can be adjusted accordingly to achieve optimal treatment outcome. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Mice lacking Gpr37 exhibit decreased expression of the myelin-associated glycoprotein MAG and increased susceptibility to demyelination.

    Science.gov (United States)

    Smith, Brilee M; Giddens, Michelle M; Neil, Jessica; Owino, Sharon; Nguyen, TrangKimberly T; Duong, Duc; Li, Fengqiao; Hall, Randy A

    2017-09-01

    GPR37 is an orphan G protein-coupled receptor that is predominantly expressed in the brain and found at particularly high levels in oligodendrocytes. GPR37 has been shown to exert effects on oligodendrocyte differentiation and myelination during development, but the molecular basis of these actions is incompletely understood and moreover nothing is known about the potential role(s) of this receptor under demyelinating conditions. To shed light on the fundamental biology of GPR37, we performed proteomic studies comparing protein expression levels in the brains of mice lacking GPR37 and its close relative GPR37-like 1 (GPR37L1). These studies revealed that one of the proteins most sharply decreased in the brains of Gpr37/Gpr37L1 double knockout mice is the myelin-associated glycoprotein MAG. Follow-up Western blot studies confirmed this finding and demonstrated that genetic deletion of Gpr37, but not Gpr37L1, results in strikingly decreased brain expression of MAG. Further in vitro studies demonstrated that GPR37 and MAG form a complex when expressed together in cells. As loss of MAG has previously been shown to result in increased susceptibility to brain insults, we additionally assessed Gpr37-knockout (Gpr37 - / - ) vs. wild-type mice in the cuprizone model of demyelination. These studies revealed that Gpr37 - / - mice exhibit dramatically increased loss of myelin in response to cuprizone, yet do not show any increased loss of oligodendrocyte precursor cells or mature oligodendrocytes. These findings reveal that loss of GPR37 alters oligodendrocyte physiology and increases susceptibility to demyelination, indicating that GPR37 could be a potential drug target for the treatment of demyelinating diseases such as multiple sclerosis. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Serological diagnosis and prognosis of severe acute pancreatitis by analysis of serum glycoprotein 2.

    Science.gov (United States)

    Roggenbuck, Dirk; Goihl, Alexander; Hanack, Katja; Holzlöhner, Pamela; Hentschel, Christian; Veiczi, Miklos; Schierack, Peter; Reinhold, Dirk; Schulz, Hans-Ulrich

    2017-05-01

    Glycoprotein 2 (GP2), the pancreatic major zymogen granule membrane glycoprotein, was reported to be elevated in acute pancreatitis in animal models. Enzyme-linked immunosorbent assays (ELISAs) were developed to evaluate human glycoprotein 2 isoform alpha (GP2a) and total GP2 (GP2t) as specific markers for acute pancreatitis in sera of 153 patients with acute pancreatitis, 26 with chronic pancreatitis, 125 with pancreatic neoplasms, 324 with non-pancreatic neoplasms, 109 patients with liver/biliary disease, 67 with gastrointestinal disease, and 101 healthy subjects. GP2a and GP2t levels were correlated with procalcitonin and C-reactive protein in 152 and 146 follow-up samples of acute pancreatitis patients, respectively. The GP2a ELISA revealed a significantly higher assay accuracy in contrast to the GP2t assay (sensitivity ≤3 disease days: 91.7%, specificity: 96.7%, positive likelihood ratio [LR+]: 24.6, LR-: 0.09). GP2a and GP2t levels as well as prevalences were significantly elevated in early acute pancreatitis (≤3 disease days) compared to all control cohorts (ppancreatitis at admission compared with mild cases (ppancreatitis with lethal outcome was 7.8 on admission (p=0.0222). GP2a and GP2t levels were significantly correlated with procalcitonin [Spearman's rank coefficient of correlation (ρ)=0.21, 0.26; p=0.0110, 0.0012; respectively] and C-reactive protein (ρ=0.37, 0.40; ppancreatitis and analysis of GP2a can aid in the differential diagnosis of acute upper abdominal pain and prognosis of severe acute pancreatitis.

  13. Molecular characterization of glycoprotein genes and phylogenetic analysis of two swine paramyxoviruses isolated from United States.

    Science.gov (United States)

    Qiao, Dan; Janke, Bruce H; Elankumaran, Subbiah

    2009-08-01

    Two swine paramyxoviruses (SPMV)-(81-19252 (Texas-81) and 92-7783 (ISU-92)-were isolated from encephalitic pigs in the United States in 1981 and 1992. Antigenic, morphologic, and biological characteristics of these two viruses were essentially similar to members of the family Paramyxoviridae. Antigenic analysis by indirect fluorescent antibody, immunoblot, and one-way cross-neutralization tests placed these viruses along with bovine parainfluenza 3 (BPIV3) viruses. Purified virions were 50-300 nm in size and morphologically indistinguishable from other paramyxoviruses. These two viruses hemagglutinated red blood cells and had neuraminidase activity. The gene junctions of fusion (F) and hemagglutinin (HN) glycoprotein genes of these viruses contained highly conserved transcription start and stop signal sequences and trinucleotide intergenic regions similar to other Paramyxoviridae. The F gene of ISU-92 was longer than Texas-81 due to insertion of a 24-nucleotide "U"-rich 3' untranslated region. Structure-based sequence alignment of glycoproteins of these two SPMVs indicated that they are essentially similar in structure and function to parainfluenzaviruses. The Texas-81 strain was closely related to BPIV3 Shipping Fever (SF) strain at nucleotide and amino acid level, while the ISU-92 strain was more closely related to BPIV3 910N strain. The envelope glycoproteins of ISU-92 had only approximately 92 and approximately 96% identity at nucleotide and amino acid levels with BPIV3-SF strain, respectively. The high sequence identities to BPIV3 indicated cross-species infection in pigs. Phylogenetic analyses based on both F protein and HN protein suggested the classification of these viruses into the subfamily Paramyxovirinae, genus Respirovirus, and genotype A of BPIV3.

  14. Ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion.

    Directory of Open Access Journals (Sweden)

    Shridhar Bale

    2011-11-01

    Full Text Available Ebolavirus belongs to the family filoviridae and causes severe hemorrhagic fever in humans with 50-90% lethality. Detailed understanding of how the viruses attach to and enter new host cells is critical to development of medical interventions. The virus displays a trimeric glycoprotein (GP(1,2 on its surface that is solely responsible for membrane attachment, virus internalization and fusion. GP(1,2 is expressed as a single peptide and is cleaved by furin in the host cells to yield two disulphide-linked fragments termed GP1 and GP2 that remain associated in a GP(1,2 trimeric, viral surface spike. After entry into host endosomes, GP(1,2 is enzymatically cleaved by endosomal cathepsins B and L, a necessary step in infection. However, the functional effects of the cleavage on the glycoprotein are unknown.We demonstrate by antibody binding and Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS of glycoproteins from two different ebolaviruses that although enzymatic priming of GP(1,2 is required for fusion, the priming itself does not initiate the required conformational changes in the ectodomain of GP(1,2. Further, ELISA binding data of primed GP(1,2 to conformational antibody KZ52 suggests that the low pH inside the endosomes also does not trigger dissociation of GP1 from GP2 to effect membrane fusion.The results reveal that the ebolavirus GP(1,2 ectodomain remains in the prefusion conformation upon enzymatic cleavage in low pH and removal of the glycan cap. The results also suggest that an additional endosomal trigger is necessary to induce the conformational changes in GP(1,2 and effect fusion. Identification of this trigger will provide further mechanistic insights into ebolavirus infection.

  15. Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture.

    Science.gov (United States)

    Effantin, Grégory; Estrozi, Leandro F; Aschman, Nick; Renesto, Patricia; Stanke, Nicole; Lindemann, Dirk; Schoehn, Guy; Weissenhorn, Winfried

    2016-07-01

    Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae.

  16. Beta2-glycoprotein I dependent anticardiolipin antibodies and lupus anticoagulant in patients with recurrent pregnancy loss.

    Directory of Open Access Journals (Sweden)

    Kumar K

    2002-01-01

    Full Text Available AIM: The present study was aimed to define the incidence of antiphospholipid antibodies of different types lupus anticoagulant (LAC, venereal disease research laboratory test (VDRL and Beta2-glycoprotein I dependent anticardiolipin antibodies Beta2 I aCL in our cohort of population experiencing recurrent pregnancy loss (RPL from Andhra Pradesh, South India. SETTING AND DESIGN: A referral case-control study at a tertiary centre over a period of 5 years. PARTICIPANTS: 150 couples experiencing 3 or more recurrent pregnancy losses with similar number of matched controls. MATERIAL AND METHODS: LAC activity was measured by the activated partial thromboplastin time (aPTT according to the method of Proctor and Rapaport with relevant modifications. VDRL analysis was performed by the kit method supplied by Ranbaxy Diagnostics Limited and Beta2 Glycoprotein I dependent anticardiolipin antibodies were estimated by ELISA kit (ORGen Tech, GmbH, Germany with human Beta2 Glycoprotein I as co-factor. STATISTICAL ANALYSIS: Statistical analysis was performed using Student′s t test. RESULTS: LAC activity was found positive in 11 women (10.28%. The mean +/- SE Beta2 I aCL concentration in the study group was 14.53 (micro/ml +/- 1.79 (range 0 to 90.4 micro/ml which was higher than the control group with a mean +/- SE of 7.26 (micro/ml +/- 0.40 (range 0 to 18 u/ml. The binding of the antibodies to the antigen was observed in 40.24% (n=33 of the cases compared to 6.09% (n=5 in controls. VDRL test was positive in 7(2.34% individuals (3 couples and 1 male partner and none among controls. CONCLUSIONS: The present study indicates the importance of antiphospholipid antibodies in women experiencing RPL and suggests the usefulness of screening for these antibodies as a mandatory routine for instituting efficient therapeutic regimens for a successful outcome of pregnancy.

  17. Genetic characterization of complete open reading frame of glycoprotein C gene of bovine herpesvirus 1

    Directory of Open Access Journals (Sweden)

    Saurabh Majumder

    2013-10-01

    Full Text Available Aim: To characterize one of the major glycoprotein genes viz., glycoprotein C (gC; UL44, unique long region 44 of bovineherpesvirus 1(BoHV1 of Indian origin at genetic and phylogenetic level.Materials and Methods: A bovine herpesvirus 1 isolate viz., (BoHV1/IBR 216 II/ 1976/ India maintained at Division ofVirology, IVRI, Mukteswar was used for the current study. The DNA was extracted using commercial kit and the completeORF of gC gene was amplified, cloned, and sequenced by conventional Sanger sequencing method. The sequence wasgenetically and phylogenetically analysed using various bioinformatic tools. The sequence was submitted in the Genbankwith accession number Kc756965.Results: The complete ORF of gC gene was amplified and sequenced. It showed 100% sequence homology with referencecooper strain of BoHV1 and divergence varied from 0% to 2.7% with other isolates of BoHV1. The isolate under study haddivergence of 9.2%, 13%, 26.6%, and 9.2% with BoHV5 (Bovine herpesvirus 5, CvHV1 (Cervid herpesvirus 1, CpHV1(Caprine herpesvirus 1, and BuHV1 (Bubaline herpesvirus 1, respectively.Conclusion: This is the first genetic characterization of complete open reading frame (ORF of glycoprotein C gene (UL44 ofIndian isolate of BoHV1. The gC gene of BoHV1 is highly conserved among all BoHV1 isolates and it can be used as a targetfor designing diagnostic primers for the specific detection of BoHV1.

  18. Anti-beta2 glycoprotein 1 and the anti-phospholipid syndrome.

    LENUS (Irish Health Repository)

    Keane, Pearse A

    2012-02-03

    PURPOSE: To describe a patient who presented with bilateral retinal vascular occlusion and the use of anti-beta2 glycoprotein 1 (GPI) antibody testing in the diagnosis of antiphospholipid syndrome. DESIGN: Observational case report. METHODS: Hematological investigations were performed on a 49-year-old man who presented with rapid onset of bilateral severe central retinal vein occlusion. RESULTS: Lupus anticoagulant and anticardiolipin antibody testing was negative. Markedly raised titers of anti-beta2 GPI antibodies were detected on two separate occasions. CONCLUSIONS: The raised titers of anti-beta2 GPI antibodies were considered to strongly suggest an underlying diagnosis of the antiphospholipid syndrome.

  19. Reduction of cerebral glucose utilization by the HIV envelope glycoprotein Gp-120

    International Nuclear Information System (INIS)

    Kimes, A.S.; London, E.D.; Szabo, G.; Raymon, L.; Tabakoff, B.

    1991-01-01

    Gp-120 is a glycoprotein constituent of the human immunodeficiency virus (HIV) envelope. The effects of gp-120 on cerebral glucose utilization in rats were studied by the quantitative 2-deoxy-D-[1-14C] glucose method. Intracerebroventricular injection of gp-120 significantly reduced glucose utilization in the lateral habenula and the suprachiasmatic nucleus and decreased the global cerebral metabolic rate for glucose. The findings suggest that gp-120 and closely related peptides can alter neuronal function, thereby contributing to the sequelae of HIV infection

  20. Anterograde glycoprotein-dependent transport of newly generated rabies virus in dorsal root ganglion neurons.

    Science.gov (United States)

    Bauer, Anja; Nolden, Tobias; Schröter, Josephine; Römer-Oberdörfer, Angela; Gluska, Shani; Perlson, Eran; Finke, Stefan

    2014-12-01

    Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 μm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 μm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 μm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of

  1. Characterization of glycoprotein biopharmaceutical products by Caliper LC90 CE-SDS gel technology.

    Science.gov (United States)

    Chen, Grace; Ha, Sha; Rustandi, Richard R

    2013-01-01

    Over the last decade, science has greatly improved in the area of protein sizing and characterization. Efficient high-throughput methods are now available to substitute for the traditional labor-intensive SDS-PAGE methods, which alternatively take days to analyze a very limited number of samples. Currently, PerkinElmer(®) (Caliper) has designed an automated chip-based fluorescence detection method capable of analyzing proteins in minutes with sensitivity similar to standard SDS-PAGE. Here, we describe the use and implementation of this technology to characterize and screen a large number of formulations of target glycoproteins in the 14-200 kDa molecular weight range.

  2. Inactivated Recombinant Rabies Viruses Displaying Canine Distemper Virus Glycoproteins Induce Protective Immunity against Both Pathogens.

    Science.gov (United States)

    da Fontoura Budaszewski, Renata; Hudacek, Andrew; Sawatsky, Bevan; Krämer, Beate; Yin, Xiangping; Schnell, Matthias J; von Messling, Veronika

    2017-04-15

    The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains. IMPORTANCE Rabies virus and canine distemper virus (CDV) cause high mortality rates and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins were protected from lethal CDV challenge, whereas all animals that received

  3. Bactericidal action of a glycoprotein from the body surface mucus of giant African snail.

    Science.gov (United States)

    Otsuka-Fuchino, H; Watanabe, Y; Hirakawa, C; Tamiya, T; Matsumoto, J J; Tsuchiya, T

    1992-04-01

    1. Bactericidal action of a glycoprotein, Achacin, purified from the giant African snail, Achatina fulica Férussac, has been studied. 2. Achacin kills both gram-positive and gram-negative bacteria, but only in their growing states. 3. Achacin does not have any bacteriolytic activity. 4. The strain which has no cell wall is a little more sensitive than the native strain and the cell membrane-damaged strain. 5. Achacin was observed on the cytoplasmic membrane and on the cell wall of treated Escherichia coli by immunoelectron microscopy. 6. Achacin attacks the cytoplasmic membrane of the cell.

  4. Clinical relevance of β₂-glycoprotein-I plasma levels in antiphospholipid syndrome (APS).

    Science.gov (United States)

    Banzato, Alessandra; Pengo, Vittorio

    2014-06-01

    Antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid (aPL) antibodies associated with thrombosis or pregnancy morbidity. The antibodies mainly involved in this disorder are directed against β2-glycoprotein I (β2-GPI). β2-GPI plasma level is usually not reported in studies on APS, because it is not regarded as relevant to the diagnosis and prognosis of APS. Nevertheless its measurement may be important for understanding the pathophysiology of the syndrome. This review summarizes available data from the literature on plasma concentrations of β2-GPI in patients with different antibody profiles.

  5. HIV envelope glycoprotein imaged at high resolution | Center for Cancer Research

    Science.gov (United States)

    The outer surface of the human immunodeficiency virus (HIV) is surrounded by an envelope studded with spike-shaped glycoproteins called Env that help the deadly virus identify, bind, and infect cells. When unbound, Env exists in a “closed” conformational state. Upon binding with target cells, such as CD4+ T cells, the protein transitions to an “open” configuration. Given that Env is the only viral protein expressed on HIV’s surface, knowing its detailed structure—especially in the unbound state—may be critical for designing antibodies and vaccines against HIV.

  6. Immune response to synthetic peptides representing antigenic sites on the glycoprotein of infectious hematopoietic necrosis virus

    Science.gov (United States)

    Emmenegger, Eveline J.; Huang, C.; LaPatra, S.; Winton, James R.

    1995-01-01

    Summary ― Monoclonal antibodies against infectious hematopoietic necrosis virus have been used to react with recombinant expression products in immunoblots and to select neutralization-resistant mutants for sequence analysis. These strategies identified neutralizing and non-neutralizing antigenic sites on the viral glycoprotein. Synthetic peptides based upon the amino acid sequences of these antigenic sites were synthesized and were injected together with an adjuvant into rainbow trout. The constructs generally failed to stimulate neutralizing antibodies in the fish. These results indicate that we need to understand more about the ability of peptide antigens to stimulate fish immune systems.

  7. Resting lymphocyte transduction with measles virus glycoprotein pseudotyped lentiviral vectors relies on CD46 and SLAM

    International Nuclear Information System (INIS)

    Zhou Qi; Schneider, Irene C.; Gallet, Manuela; Kneissl, Sabrina; Buchholz, Christian J.

    2011-01-01

    The measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors were found to be essential.

  8. Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells

    Energy Technology Data Exchange (ETDEWEB)

    Rusche, J.R.; Lynn, D.L.; Robert-Guroff, M.; Langlois, A.J.; Lyerly, H.K.; Carson, H.; Krohn, K.; Ranki, A.; Gallo, R.C.; Bolognesi, D.P.; Putney, S.D.

    1987-10-01

    The human immunodeficiency virus envelope gene was expressed in insect cells by using a Baculovirus expression vector. The protein has an apparent molecular mass of 160 kDa, appears on the surface of infected insect cells, and does not appear to be cleaved to glycoproteins gp120 and gp41. Goats immunized with the 160-kDa protein have high titers of antibody that neutralizes virus infection as measured by viral gene expression or cell cytolysis. In addition, immune sera can block fusion of human immunodeficiency virus-infected cells in culture. Both neutralization and fusion-blocking activities are bound to and eluted from immobilized gp120.

  9. Ligand and structure-based classification models for Prediction of P-glycoprotein inhibitors

    DEFF Research Database (Denmark)

    Klepsch, Freya; Poongavanam, Vasanthanathan; Ecker, Gerhard Franz

    2014-01-01

    The ABC transporter P-glycoprotein (P-gp) actively transports a wide range of drugs and toxins out of cells, and is therefore related to multidrug resistance and the ADME profile of therapeutics. Thus, development of predictive in silico models for the identification of P-gp inhibitors is of great......Score resulted in the correct prediction of 61 % of the external test set. This demonstrates that ligand-based models currently remain the methods of choice for accurately predicting P-gp inhibitors. However, structure-based classification offers information about possible drug/protein interactions, which helps...

  10. In Vivo Role of Complement-Interacting Domains of Herpes Simplex Virus Type 1 Glycoprotein Gc

    OpenAIRE

    Lubinski, John; Wang, Liyang; Mastellos, Dimitri; Sahu, Arvind; Lambris, John D.; Friedman, Harvey M.

    1999-01-01

    Immune evasion is critical for survival of viruses that establish persistent or recurrent infections. However, at the molecular level, little is known about how viruses evade immune attack in vivo. Herpes simplex virus (HSV)-1 glycoprotein gC has two domains that are involved in modulating complement activation; one binds C3, and the other is required for blocking C5 and properdin (P) binding to C3. To evaluate the importance of these regions in vivo, HSV-1 gC mutant viruses were constructed ...

  11. Herpes Simplex Virus Type 1 Glycoprotein gC Mediates Immune Evasion In Vivo

    OpenAIRE

    Lubinski, John M.; Wang, Liyang; Soulika, Athena M.; Burger, Reinhard; Wetsel, Rick A.; Colten, Harvey; Cohen, Gary H.; Eisenberg, Roselyn J.; Lambris, John D.; Friedman, Harvey M.

    1998-01-01

    Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact a...

  12. Chimeric Bovine Respiratory Syncytial Virus with Attachment and Fusion Glycoproteins Replaced by Bovine Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase and Fusion Proteins

    Science.gov (United States)

    Stope, Matthias B.; Karger, Axel; Schmidt, Ulrike; Buchholz, Ursula J.

    2001-01-01

    Chimeric bovine respiratory syncytial viruses (BRSV) expressing glycoproteins of bovine parainfluenza virus type 3 (BPIV-3) instead of BRSV glycoproteins were generated from cDNA. In the BRSV antigenome cDNA, the open reading frames of the major BRSV glycoproteins, attachment protein G and fusion protein F, were replaced individually or together by those of the BPIV-3 hemagglutinin-neuraminidase (HN) and/or fusion (F) glycoproteins. Recombinant virus could not be recovered from cDNA when the BRSV F open reading frame was replaced by the BPIV-3 F open reading frame. However, cDNA recovery of the chimeric virus rBRSV-HNF, with both glycoproteins replaced simultaneously, and of the chimeric virus rBRSV-HN, with the BRSV G protein replaced by BPIV-3 HN, was successful. The replication rates of both chimeras were similar to that of standard rBRSV. Moreover, rBRSV-HNF was neutralized by antibodies specific for BPIV-3, but not by antibodies specific to BRSV, demonstrating that the BRSV glycoproteins can be functionally replaced by BPIV-3 glycoproteins. In contrast, rBRSV-HN was neutralized by BRSV-specific antisera, but not by BPIV-3 specific sera, showing that infection of rBRSV-HN is mediated by BRSV F. Hemadsorption of cells infected with rBRSV-HNF and rBRSV-HN proved that BPIV-3 HN protein expressed by rBRSV is functional. Colocalization of the BPIV-3 glycoproteins with BRSV M protein was demonstrated by confocal laser scan microscopy. Moreover, protein analysis revealed that the BPIV-3 glycoproteins were present in chimeric virions. Taken together, these data indicate that the heterologous glycoproteins were not only expressed but were incorporated into the envelope of recombinant BRSV. Thus, the envelope glycoproteins derived from a member of the Respirovirus genus can together functionally replace their homologs in a Pneumovirus background. PMID:11533200

  13. Role of the Conserved Ologomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast Cancer Cells

    National Research Council Canada - National Science Library

    Zolov, Sergey

    2004-01-01

    .... We propose that the COG3 protein plays one of the main roles in these processes. We utilized RNA interference assay to knockdown COG3p in HeLa cells to determine the effect of its depletion on Golgi proteins localization...

  14. Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GP1 Ectodomain Shedding

    Science.gov (United States)

    2008-12-23

    This interaction should be susceptible to disruption by treatment with ani- onic detergents commonly used in PAGE buffers. How- ever, our Western blot...Department of Health and Human Services/ National Institutes of Health/National Institute of Allergy and Infectious Diseases Challenge and Partnership Grant

  15. Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GPI Ectodomain Shedding

    Science.gov (United States)

    2008-12-23

    with ani- onic detergents commonly used in PAGE buffers. How- ever, our Western blot detection of a prominent high molecular component purified from...National Institute of Allergy and Infectious Diseases Challenge and Partnership Grant Number 1 UC1 AI067188-01, in association with USAMRIID Military

  16. Dystrophin insufficiency causes selective muscle histopathology and loss of dystrophin-glycoprotein complex assembly in pig skeletal muscle

    Science.gov (United States)

    Duchenne muscular dystrophy (DMD) is caused by a dystrophin deficiency while Becker muscular dystrophy (BMD) is caused by a dystrophin insufficiency or expression of a partially functional protein product. Both of these dystrophinopathies are most commonly studied using the mdx mouse and a golden r...

  17. An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

    International Nuclear Information System (INIS)

    Tiwari, Vaibhav; Darmani, Nissar A.; Thrush, Gerald R.; Shukla, Deepak

    2009-01-01

    Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

  18. The hypobranchial mucin of the whelk Buccinum undatum L. Properties of the mucin and of the glycoprotein component.

    Science.gov (United States)

    Hunt, S; Jevons, F R

    1965-12-01

    1. The composition of the hypobranchial mucin from Buccinum undatum is reported. 2. The amino acid composition was determined; aspartic acid and glutamic acid contribute almost 24% of the total amino acids in the mucin. 3. Serine, threonine and alanine, in the proportions 2:1:1 respectively, were detected as N-terminal residues, implying the presence of at least four protein chains. 4. A glycoprotein component was isolated by phenol precipitation. 5. The glycoprotein contained 8% of neutral sugars comprising glucose, galactose, mannose and fucose, and 4.5% of hexosamine, comprising glucosamine and galactosamine in equal proportions. 6. A method is described for the preparation of glycopeptides from the glycoprotein. 7. The comparative biochemistry of the mucin is discussed.

  19. Mapping of surface glycoproteins of Trypanosoma cruzi by two-dimensional electrophoresis. A correlation with the cell invasion capacity.

    Science.gov (United States)

    Andrews, N W; Katzin, A M; Colli, W

    1984-05-02

    The cell-surface iodinatable proteins of Trypanosoma cruzi have been analyzed by two-dimensional polyacrylamide gel electrophoresis under equilibrium conditions. Antigenic polypeptides were characterized after immunoprecipitation and glycoproteins were identified by means of lectin-affinity chromatography. Two glycoproteins, with affinity for concanavalin A, were found to be common to both infective (trypomastigote) and non-infective (epimastigote) forms: protein 1 (90 kDa, pI 5.5-6.5) and protein 2 (80 kDa, pI 5.3-6.3). In epimastigotes a specific concanavalin-A-binding surface glycoprotein (70 kDa, pI 5.5) was identified. Trypomastigote forms, on the other hand, presented several specific iodinatable surface components: glycoproteins 3(85 kDa, pI 5.5), 4 (85 kDa, pI 5.0), 6 (100 kDa, pI 6.5), 7 (120 kDa, pI 6.3), 8 (68 kDa, pI 6.7) and several minor high-molecular-mass acid proteins, all containing glucose and/or mannose, and glycoprotein 5 (85 kDa, pI 6.3-7.5), containing N-acetyl-D-glucosamine (Tc-85). Proteins 1, 2 and 5 were the only ones which gave clear evidence of charge heterogeneity. Most of the surface proteins of trypomastigote forms, the exception being proteins 3, 4 and 8, were removed by treatment with trypsin. This proteolytic treatment results in 90% inhibition of the in vitro vertebrate-cell-invasion capacity of the parasites. Upon reincubation in culture medium for 4 h, the trypsin-removed glycoproteins are again detected, an observation that correlates well with the recovery of the cell-penetration capacity observed in the same period.

  20. Synthesis of fucosyl-containing glycoproteins of the vitelline coat in oocytes of Ciona intestinalis (Ascidia).

    Science.gov (United States)

    Rosati, F; Cotelli, F; De Santis, R; Monroy, A; Pinto, M R

    1982-01-01

    The sperm receptors of the ascidian oocyte are located at the outer surface of the vitelline coat (formerly called the chorion). The fucose residues are the receptor's most important components for sperm recognition and binding. We asked whether the fucosyl-containing glycoproteins of the vitelline coat are a product of the oocyte, the follicle cells, or the test cells. Ovaries of Ciona intestinalis were injected with L-[3H]fucose and the progress of its incorporation was followed by using autoradiography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the injected gonads and of the isolated vitelline coats. We found that incorporation of fucose begins within the vitellogenic oocytes, and fucose slowly accumulates in the differentiating vitelline coat. At no time could fucose incorporation be detected in the follicle cells or in the test cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of vitelline coats prepared from the injected ovaries showed fucose incorporation into the same three glycoproteins present in vitelline coats from mature oocytes and identified by their affinity for 125I-labeled fucose-binding protein [Pinto, M. R., De Santis, R., D'Alessio, G. & Rosati, F. (1981) Exp. Cell Res. 132, 289-295]. A radioactive band not found in the mature oocyte was also present. Images PMID:6952240